University of Southern California Dissertations and Theses

Studies on the bacterial oxidation of saturated fatty acids and their derivatives

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Studies on the bacterial oxidation of saturated fatty acids and their derivatives

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Content STUDIES O N TBE BACTERIAL OXIDATION OP SATURATED PATTY ACIDS AND THEIR DERIVATIVES A D i s s e r t a t i o n P r e s e n te d t o th e P a c u lt y o f t h e O rad u ate S c h o o l U n iv e r s it y o f S o u th e r n C a lif o r n ia I n p a r t i a l P u l f i lim e n t o f t h e R e q u irem en ts f o r th e D eg ree D o c to r o f P h ilo s o p h y by D a n ie l I v l e r June 1954 PK. D BA. 5-4 T ’ ^ 5 This dissertation, written by p ^ i e l Ivl^ under the direction ofl^.^.Guidance Committee, and approved by all its members, has been pre sented to and accepted by the Faculty of the Graduate School, in partial fulfillment of re quirements for the degree of D O C T O R OF P H I L O S O P H Y Date. Committee on Studies Chairman V Dean A C E N O W I E D G m m N T j I w is h t o e x p r e s s my s i n c e r e a p p r e c ia t io n t o th e | i members o f my g u id a n c e c o m m itte e , D r s . M. D . A pplem an, J . W. B arth olom ew , H. M # K u r tz , W * M arx, and to D ean H. J . D e u e l f o r t h e i r c o n s t r u c t iv e c r i t i c i s m and g e n e r o u s a d v ic e .j My th a n k s a l s o t o f e l l o w s tu d e n t, Mr. J . A . L a z a r o n i, J r . , f o r t h e n it r o g e n d e t e r m in a t io n s , and my c o ll e a g u e . D r . J . B . W o lfe , f o r h i s c o ll a b o r a t io n i n v a r io u s r e s e a r c h e n d e a v o r s . I s h a l l a lw a y s b e in d e b te d t o D r . S . C. R it t e n b e r g f o r th e h ig h q u a l i t y o f h i s g u id a n c e , f o r h i s sound l o g i c , and f o r h i s s t im u la t in g v i s i o n w h ich h a s b een a c o n s t a n t s o u r c e o f i n s p i r a t i o n . To my w if e and f a m ily I owe an u n su rm o u n ta b le d e b t f o r t h e i r p a t ie n c e and u n d e r s ta n d in g . TABm OF COHTEUTS CaAFTER PAGE I . lîîTRODUGTIGSr................................................................................... 1 The p rob lem .......................... 9 I I . THE OXIDATION O F PHENYL SUBSTITUTED PATTY ACIDS BY RESTING CELLS OF VARIOUS BACTERIA . . . . 11 I n t r o d u c t io n . . . . . . .. 11 M a t e r ia ls and m eth ods . . . . . . . . . . . . 11 E x p e r im e n ta l r e s u l t s . . . . . . . . . 15 O x id a tio n p a t t e r n s . . . . . . . . . . 15 A d a p ta tio n e x p e r im e n ts . . . . . . . . . . 15 D i s c u s s i o n .......................... . . . . . . . . . . . 25 Summary . . . . . . . . .......................... . . . . . 29 I I I . THE OXIDATION OF ALIPHATIC PATTY ACIDS BY CELL- FREE SYSTEMS. FROM SERRATlA MARCESCENS ( A L P H I N ) ............................................. 52 I n t r o d u c t io n . . . . . .................................................... 5 2 M ethods . . . . . . . . . . . . . . . . . 55 P r e p a r a tio n o f c e l l - f r e e e x t r a c t s . . . . . 55 M anom etric d e te r m in a tio n . . . . . . . . . 54 p r o d u c t a n a l y s i s .. .. .. .. ... 54 E x p e r im e n ta l r e s u l t s ..................... . . .. . .. 55 C o fa c to r r e q u ir e m e n ts . . . . . . . . . . . 55! P ro d u ct a n a l y s i s . . . . . . . . . . . . . 59 V CHAPTER page S u m m ary......................... 41 IV . THE OXIDATION OP ALIPHATIC FATTY ACIDS BY CELL- FREE SYSTEMS FROM PSEUDOMONAS FLUORESCENS P .6 . (1 ) PRELIMINARY S T U D IE S .................... 4 3 I n t r o d u c t i o n ............................................................ 43 M e t h o d s ............................... ..... . .......................................... 43 P r e p a r a tio n o f c e l l - f r e e e x t r a c t . . . . . 44 E x p e r im e n ta l r e s u l t s ............................................... 45 C o fa c to r r e q u i r e m e n t s ................................... 45 E s s e n t i a l n a tu r e o f coenzym e A and ATP • . 47 D e t e c t io n o f a c e t a t e .......................... 5 0 Summary and d i s c u s s i o n . . . . . . . . . . . 5 2 V . THE OXIDATION OP ALIPHATIC FATTY ACIDS BY CELL- FREE SYSTEMS PROM PSEUDOMONAS FLUORES CENS P . 6 (2 ) FRACTIONATION O P CELL-FREE EXTRACTS AND PROPERTIES OF THE PARTIALLY PURIFIED ENZYME SYSTEM ............................................. 5 3 I n t r o d u c t io n ......................................................... 5 3 M ethods .................................................................. 5 3 E x p e r im e n ta l r e s u l t s ................................... 6 3 Ammonium s u l f a t e f r a c t i o n a t i o n . . . . . 5 3 O x id iz in g c a p a c it y o f t h e S40 - 6 O f r a c t i o n 56 Vi CHAPTER PAGE M agnesium e f f e c t on a d i a l y s e d 840-60 f r a c t i o n ........................................................................ 6 0 S u m m ary............................................................................................ 6 2 V I. THE OXIDATION OF ALIPHATIC PATTY ACIDS BY CELL- FREE SYSTEMS PROM PSEUDOMONAS PUJORSSCEMS P .6 . ( 3 ) QUALITATIVE AM D QUANTITATIVE TRAP PING OF ACTIVATED CAPRATE . . . 64 I n t r o d u c t io n « ..................... « .........................................« 64 M ethods . . . . . . . . . . . .............................. .. 64 E x p e r im e n ta l r e s u l t s . . . . . . . 65 V I I . THE OXIDATION OF ALIPHATIC FATTY ACIDS BY CELL- FREE SYSTEMS FROM BACTERIA. DISCUSSION OF RESULTS............................................................................................. 68 V I I I . R E S U M E ................................................................................................. 74 BIBLIOGRAPHY . .............................. 79 LIST O P TABLES TABLE PAGE I , O x id a tio n o f P h e n y l S u b s t it u t e d P a t t y A c id s by F iv e B a c t e r ia ..................................... 1 4 ' I I * O xygen U p tak e on V a r io u s P h en y l S u b s t it u t e d j F a t t y A c id s by t h e S o i l i s o l a t e S *51 . . . . 19 I I I * Oxygen U p take on V a r io u s P h e n y l S u b s t it u t e d P a t t y A c id s by T h ree Pseudom onads and S . M arce s c e n s (A lp h in ) .......................... • • • • • • • 22 IV* O x id a tio n o f c a p r a te and C a p r y la te by Crude C e ll- F r e e E x t r a c t s o f S* M a rcescen s (A lp h in ) i n th e P r e s e n c e and A b sen ce o f a C o fa c to r S u p p l e m e n t ......................... ........................................................ 37 V* C o fa c to r E f f e c t on C a p ra te O x id a tio n w it h Crude C e ll- F r e e E x t r a c t s o f S* M a rcescen s (A lp h in ) . * ......................................... « .............................. 38 VI* R f v a lu e s o f C a p r y la te O x id a tio n P r o d u c ts . . . 40 V II* O x id a tio n o f C ap rate and A c e t a t e by Crude C e l l - F r e e E x t r a c t s o f P* F lu o r e s c e n s P .6 i n th e P r e s e n c e and A b se n c e o f a C o fa c to r S u p p le m ent ......................................................... . . . . . . * * 46 V I I I . E f f e c t o f ATP and CoA on C ap rate O x id a tio n by a Crude C © ll* F ree E x t r a c t o f P . F lu o r e s c e n s . 48 v î i i TABLE PAGE : IX . E f f e c t o f GoA on C ap rate O x id a tio n by a D ow ex- j 1 - H y d r o c h lo r id e T r e a te d C e ll- F r e e E x tr a c t o f P , F lu o r e s c e n s P .6 . . . . . . . . . . 4 9 X . E f v a lu e s o f C a p ra te O x id a tio n P r o d u c ts o f Crude C e ll- F r e e E x t r a c t s o f p . F lu o r e s c e n s P .6 . . . .................................................................................. 5 1 X I . C a p ra te O x id a tio n by R e d is s o lv e d Ammonium S u l f a t e P r e c i p i t a t e d F r a c t io n s o f a C e ll- F r e e E x t r a c t o f P . F lu o r e s c e n s , p .6 . . . . . . . 55 X I I . O x id iz in g C a p a c ity o f t h e S40 - 6 O F r a c t io n . . . 57 X I I I . The E f f e c t o f F lu o r id e on C ap rate O x id a tio n by t h e S40 - 6 O F r a c t io n . . . . . . . . . . . . . 59 X IV . R j^ v a lu e s o f H yd roxylam in e T rapped B i o l o g i c a l E s t e r s . 66 XV. Q u a n t it a t iv e D e te r m in a tio n o f B i o l o g i c a l l y Formed C ap ryl CoA . . . . . . . . . . . . . . 67 LIST OP FIGURES FIGURE PAGE 1 . O x id a tio n o f p h e n y la c e t a t e by s o i l I s o l a t e S . 6 1 . 16 2 . O x id a tio n o f P h e n y lb u ty r a te by S o i l I s o l a t e S . 5 1 . 17 3 . O x id a tio n o f P h e n y lh e p t y la t e and B e n z o a te by P . F lu o r e s c e n s A 3 .1 7 .............................................. 21 4 . O x id a tio n o f P h e n y lc a p r a te and P h e n y la c e ta te by S . M a rcescen s (A lp h in ) . . . . . . . . . . . . 24 5 . S c h e m a tic D iagram o f P h e n y l P a t t y A c id O x id a tio n b y F iv e B a c t e r ia .............................. 30 6 . E f f e c t o f M agnesium on C ap rate O x id a tio n by a D ia ly z e d 84 0 -5 0 F r a c t io n ................................... 61 7 . S c h e m a tic D iagram o f F a t t y A c id O x id a tio n by C e ll- F r e e S y s te m s . from 8 . M arcescen s (A lp h in ) and P . F lu o r e s c e n s P . 6.......................................... 73 CHAPTER I INTRODUCTION An u n d e r s ta n d in g o f th e d e t a i l e d b io c h e m ie a l e v e n ts in th e m e ta b o lism o f f a t t y a c id s b e a r s a d i r e c t r e l a t i o n t o th e i s o l a t i o n , p u r i f i c a t i o n and s t u d y o f t h e enzym es i n v o lv e d # T h is ty p e o f a p p ro a ch w it h c a r b o h y d r a te s culm in ated i n t h e e s t a b lis h m e n t o f t h e g l y c o l y t i c pathw ay s i n c e h i g h ly p u r i f i e d p r e p a r a tio n s h a v e b e e n a v a i l a b l e f o r many y e a r s t o i n v e s t i g a t o r s i n t e r e s t e d i n c a r b o h y d r a te m eta b o lism # U n t i l v e r y r e c e n t l y , in fo r m a tio n on t h e a c t u a l m echanism o f f a t t y a c id o x id a t io n h a s b een som ewhat m eager d e s p i t e a v o lu m in o u s l i t e r a t u r e on f a t t y a c id m e ta b o lis m . Many r e v ie w s can b e c o n s u lte d f o r th e e a r l y work c o n c e r n e d w it h f a t t y a c id o x i d a t i o n by a n im a l sy s te m s ( B r e u sc h , 1948; S t a d i e , 1 9 4 5 ; L e h n in g e r , 1 9 5 0 , 1 9 6 2 )# Munoz and L e l o i r (1 9 4 5 ) w ere t h e f i r s t t o o b t a in f a t t y a c id o x id a t io n b y c e l l - f r e e sy ste m s# They o b ta in e d a p a r t i c u l a t e f r a c t i o n by c e n t r i f u g i n g g u in e a p ig l i v e r h om ogen ates t h a t c a r r ie d o u t f a t t y a c id o x id a t io n when su p p lem en ted w it h ATP and o th e r c o f a c t o r s # S im ila r p r e p a r a t io n s w ere o b ta in e d by L eh n in g er (1 9 4 5 ) from r a t l i v e r # He p o s t u la t e d a f a t t y a c id p h o sp h a te e s t e r a s t h e p rim ary a c t i v e compound i n f a t t y a c id o x id a tio n # G r a f f l i n and T ! G reen ( 1 9 4 8 ) , c h e l d e l i n and B e in e r t (1 9 5 2 ) and o t h e r s a l s o i n v e s t i g a t e d f a t t y a c id o x id a t io n b y c y c lo p h o r a s e p r e p a r a t i o n s o b ta in e d fro m l i v e r and k id n e y , and o th e r sy s te m s b u t w ere u n s u c c e s s f u l i n e lu c i d a t i n g th e k e y s t e p s i n f a t t y a c id m e ta b o lis m . T h ree g e n e r a l t h e o r i e s f o r th e o x id a t iv e m echanism had d e v e lo p e d a s a r e s u l t o f i n v e s t i g a t i o n p r i o r t o 1 9 5 5 ; b e ta o x id a t io n a s p r o p o sed by Knoop ( 1 9 0 4 ) , th e m u lt ip le a l t e r n a t e o x id a t io n m echanism o f J o w e tt and Q u a s te l ( 1 9 3 5 ) , and omega o x id a t io n (V erk ade and van d er L e e , 1 9 3 2 )• By 1953 h o w ev er, b e ta o x id a t io n w as g e n e r a l ly a c c e p te d a s th e m ajor m echanism a lth o u g h t h e d e t a i l e d in te r m e d ia r y s t e p s i n f a t t y a c id o x id a t io n w ere unknown. The e l u c i d a t i o n o f th e m echanism o f b e t a o x id a t io n becam e p o s s i b l e w i t h t h e d i s c o v e r y o f OoA^ by Lipmann (1 9 5 0 , 1 9 5 3 ) and th e i d e n t i f i c a t i o n o f a c e t y l OoA a s th e a c e t y l t h i o e s t e r o f CoA by Lynen e t a l . ( 1 9 5 1 ) . N ot o n ly The f o l lo w in g a b b r e v ia t io n s a re u se d th r o u g h o u t th e d i s s e r t a t i o n : a c e t y l coA ( a c e t y l coenzym e A ) , ATP ( a d e n o s in e t r ip h o s p h a t e ) , GSH (r e d u c e d g l u t a t h i o n e ) , CoA (coen zym e A ) , DPN ( d ip h o s p h o p y r id in e n u c l e o t i d e ) , TPN ( t r ip h o s p h o p y r id in e n u c l e o t i d e ) , R -5 -P ( r i b o f l a v i n e - 5 - p h o s p h a te ), PAD ( f l a v i n e a d e n i n e d in u c le o t i d e ) , S4 0 -50 ( r e d i s s o l v e d p r e c i p i t a t e o b ta in e d a t 4 0 -6 0 p er c e n t am monium s u l f a t e s a t u r a t i o n ) , ATPase (a d e n o s in e t r i p h o s p h a t a s e ) , AMP (a d e n o s in e m o n o p h o sp h a te), PP, ( in o r g a n ic p y r o p h o s p h a te ). ----------------------------------------- — -------------------------------------------------------------------------------- " s i was t h e e l u s i v e » tw o -c a r b o n fr a g m e n t' i d e n t i f i e d b u t a new ty p e o f e n e r g y r i c h bond w as found* On th e b a s is o f t h e f i n d in g s o f Lipm ann and o f L ynen, W einhouse (1 9 5 2 ) , w ith o u t p r o v id in g e x p e r im e n ta l d a t a , p o s t u la t e d a m echanism f o r th e o x id a t io n o f f a t t y a c id s i n w h ic h th e i n i t i a l r e a c t i o n was t h e - e s t é r i f i c a t i o n o f t h e f a t t y a c id w it h c o enzyme. A . I t was assum ed t h a t t h i s a c y l f a t t y a c id w o u ld e n o l i z e t o form a m o le c u le i n w h ic h th e b e t a carb on i s h ig h ly , a c t i v a t e d by i t s a d ja c e n c y t o a d o u b le bonded ca rb o n and a d r iv in g f o r c e i s th u s p r o v id e d f o r a s p e c i f i c o x i d a t i v e a t t a c k a t t h e b e ta p o s i t i o n . The m o le c u le w ould th e n u n d erg o o x id a t io n t o form t h e b e ta k e t o coA d e r i v a t i v e . The a u th o r th e n p o s t u la t e d a t h i o - l y t i c c le a v a g e w ith t h e fo r m a tio n o f a c e t y l CoA and th e n e x t lo w e r h om ologou s f a t t y a c id e s t e r o f €oA . Su ch a m echanism y i e l d s t h e a c t i v a t e d lo w e r f a t t y a c id w ith o u t fo r m a tio n o f a f r e e f a t t y a c id a s s u c h . The a b s e n c e o f a f r e e in t e r m e d ia t e was p r e v io u s ly s u g g e s t e d by Lynen e t a l . (1 9 5 1 ) and a lm o s t s im u lt a n e o u s ly by Lardy ( 1 9 5 2 ) . The a c t i v a t i o n h y p o t h e s is was a l s o n o t new , s i n c e t h e work o f K ennedy and B ark er (1 9 5 1 ) had p r e v io u s ly shown t h e fo r m a tio n o f a c y l €oA e s t e r s d u r in g o x id a t io n o f lo w e r f a t t y a c id s by C lo s tr id iu m k l u y v e r i . A l s o , an a c t i v a t i o n o f f a t t y a c i d s by a n im a l t i s s u e s had b e e n r e p o r te d by 4 K ennedy and L e h n in g er ( 1 9 5 2 ) . A t t e n t i o n was th e n d i r e c t e d t o t h e i n i t i a l a c t i v a t i o c r e a c t i o n . M ah ler e t a l . (1 9 5 3 ) r e p o r t e d on th e p u r i f i c a t i o n and p r o p e r t i e s o f an a c t i v a t i n g enzym e i s o l a t e d from a n im a l t i s s u e s . The enzym e, term ed f a t t y a c id a c t i v a t i n g enzym e, c a t a ly z e d th e fo r m a tio n o f a c y l GoA from a w id e v a r i e t y o f f a t t y a c id s (C3 t o G^g) and v a r io u s f a t t y a c id d e r i v a t i v e s i n th e p r e s e n c e o f ATP and CoA w ith th e f o r m a tio n o f AMP and PP i n s t o i c h i o m e t r ic a m o u n ts. The r e v e r s i b i l i t y o f t h e r e a c t i o n was a l s o show n. The e q u i l i b riu m c o n s t a n t f o r t h i s r e a c t i o n w as shown t o be c l o s e t o u n i t y th u s e s t a b l i s h i n g t h a t th e a c y l CoA d e r i v a t i v e s a r e h ig h e n e r g y com pounds. A n a lo g o u s r e a c t i o n s by d i f f e r e n t a c t i v a t i n g enzym es h ave a l s o b e e n r e p o r te d f o r a c e t a t e (Lipm ann e t a l , , 1 9 5 3 ) and h ig h e r f a t t y a c id s (K ornberg and P r i c e r , 1 9 5 3 ) . W ith t h e e s ta b lis h m e n t o f a c y l CoA d e r i v a t i v e s o f th e f a t t y a c id s a s t h e t r u e s u b s t r a t e s o f t h e f a t t y a c id o x i d i z i n g e n z ^ e s , th e a c t u a l s t e p s i n th e o x id a t io n w ere d e te r m in e d a f t e r th e i s o l a t i o n i n a h i g h ly p u r i f i e d fo rm , o f t h e enzym es i n v o lv e d . M ahler (1 9 5 3 a ) d e m o n str a te d t h a t th e f i r s t s t e p i n b u t y r y l CoA o x id a t io n i s a f l a v i n e d ep en d en t d e h y d r o g e n a tio n a t th e a lp h a b e ta p o s i t i o n w it h t h e fo r m a tio n o f b u te n o y l CoA. F o llo w in g th e u n s a t u r a t io n . 5 h y d r a tio n and d e h y d r o g e n a tio n o c c u r , th e l a t t e r b e in g DPN d e p e n d e n t, w it h t h e fo r m a tio n o f th e b e ta k e t o d e r i v a t i v e , a c e t o a c e t y l CoA (L ynen and O choa, 1 9 5 3 ) . S t u d ie s w it h c a p r o a te d e m o n str a te d th e m echanism o f o x id a t io n t o b e i d e n t i c a l t o t h a t o f b u t y r a t e h o w ev e r, th e d e h y d r o g e n a se in v o lv e d e x h ib it e d a d i f f e r e n t s p e c i f i c i t y th a n t h e b u t y r y l CoA d e h y d r o g e n a s e . F u r th e r w ork by G reen e t a l , (1 9 5 3 b ) a l s o show ed t h a t th e r e a r e s e v e r a l a c t i v a t in g en zy m es, t h e s p e c i f i c i t y o f e a c h b e in g d e te r m in e d b y t h e c h a in le n g t h o f t h e f a t t y a c i d , Lynen e t a l . (1 9 5 2 ) and l a t e r S t e r n e t a l . (1 9 5 3 ) and G reen e t a l . (1 9 5 3 a ) e s t a b l i s h e d th e l a s t in te r m e d ia r y s t e p i n o x i d a t i o n a s b e in g a t h i o l y t i c c le a v a g e o f t h e b e t a k e t o GoA f a t t y a c i d . The enzym e, b e ta k e to t h i o l a s e , in , t h e p r e s e n c e o f GoA c le a v e s t h e k e to CoA compound fo rm in g a c e t y l CoA and an a c t i v a t e d f a t t y a c id o f two l e s s c a r b o n s . The o v e r a l l m echanism o f f a t t y a c id o x id a t io n e s t a b l i s h e d by t h e s e w o rk ers may b e sum m arized i n th e f o l lo w in g m anner: RGHgCHaCHaCOOH |A T P , C O A RCHaCHBOHaCOGoA |pA D RCHgCH = CH CO CO A RCHgCHOHGHgCOCoA DPN r RCHsCOCHgCOCoA CoA RGHgCOCoA + CH3COC0A p r e v io u s t o and c o n c u r r e n t ly w ith , t h e s t u d i e s on a n im a l s y s t e m s . B ark er and h i s cow ork ers d is c o v e r e d a u n iq u e mode o f f a t t y a c id m e ta h o lis m i n t h e a n a e r o b ic b a c te r iu m , £ l . k l u y v e r i . I t w as show n t h a t t h i s o r g a n ism o b t a in s e n e r g y from th e s y n t h e s i s o f f a t t y a c id s (B a r k e r , 1 9 5 7 ) by c o n v e r t in g tw o -c a r b o n compounds ( e t h a n o l and a c e t a t e ) t o f a t t y a c id s o f fo u r and s i x c a r b o n s (B o rn st e i n and B a r k e r , 1 9 4 8 a , 1 9 4 8 b ). T r a c e r e x p e r im e n ts r e v e a le d t h a t t h e c o n v e r s io n i s a n o x id a t io n - r e d u c t io n p r o c e s s i n w h ic h e t h a n o l i s o x id iz e d t o a tw o -c a r b o n compound ( a c t i v e a c e t a t e ) t h a t i s i n e q u ilib r iu m w it h a c e t a t e . The a c t i v e a c e t a t e i s c o n d e n sed w it h a c e t a t e t o a fo u r -c a r b o n compound t h a t s e r v e s a s an o x id a n t f o r th e r e a c t i o n and i s r e d u c e d 7 t o b u t y r a t e ; i n a s i m i l a r m anner, t h e a c t i v e a c e t a t e d e r iv e d from a n o th e r m o le c u le o f e th a n o l may c o n d e n se w it h b u ty r a te t o form c a p r o a te (B a rk er e t a l . , 1 9 4 5 ) . C e l l - f r e e p r e p a r a tio n s o f t h e o r g a n ism (sta d tm a n and B a r k e r, 1 9 4 9 a ) u n d er a n a e r o b ic c o n d it io n s q u a n t i t a t i v e l y c o n v e r t e th a n o l and a c e t a t e t o b u t y r a t e . A e r o b ic a lly w it h oxy g en a s e l e c t r o n a c c e p t o r , e th a n o l i s o x id iz e d t o a c e t y l p h o sp h a te and a c e t a t e , A c o n d e n s a t io n o f a c e t y l p h o sp h a te and a c e t a t e w as c a t a ly z e d by t h e c e l l - f r e e sy s te m and t h e p r o d u c t r ed u c ed t o b u t y r a t e , A c o ta ld e h y d e p ro v ed t o be an in t e r m e d ia t e i n t h e c o n v e r s io n o f e th a n o l t o a c e t y l p h o s p h a t e . The f a c t t h a t a c e t y l p h o sp h a te a p p ea red t o p a r t i c i p a te d i r e c t l y i n th e r e a c t i o n becam e an o b s t a c l e i n t h e a c c e p ta n c e o f t h e C l, k l u y v e r i sy s te m a s b e a r in g on t h e c o r r e s p o n d in g - r e a c t io n s i n a n im a ls s i n c e t h i s compound i s n e i t h e r form ed n o r u t i l i z e d by th e a n im a l t i s s u e . The r o l e o f a c e t y l p h o sp h a te w as r e s o l v e d by S ta d tm a n , N o v e l l i and Lipmann (1 9 5 1 ) when i t was shown t h a t t h i s b a c te r iu m , a s w e l l a s c e r t a i n o t h e r s , c o n t a in s th e enzym e p h o s p h o tr a n s - a c e t y l a s e w h ich i s . l a c k i n g i n a n im a l t i s s u e s , P h o sp h o - t r a n s a e e t y l a s e c a t a l y z e s r e v e r s i b l y th e t r a n s f o r m a tio n o f a c e t y l p h o sp h a te and GoA t o a c e t y l CoA and p h o sp h o r ic a c i d . The a c t u a l c o n d e n s in g com pounds i n f a t t y a c id s y n t h e s i s by 8 C l , k lu y v e r i w e re shown t o be a c e t y l CoA and a c e t a t e . The m echanism o f t h e s y n t h e s i s o f th e f a t t y a c id s was th e n r e - s t u d ie d and t h e f o l lo w in g r e v e r s i b l e r e a c t i o n se q u e n c e was a d v a n ced : A c e t y l CoA + b u ty r a t e b u t y r y l CoA + a c e t a t e É a c e t a t e A c e t y l GoA + v i n y l a c e t a t e v i n y l a c e t y l CoA b e ta h y d r o x y b u ty r y 1 GoA t CoA 2 a c e t y l GoA a c e t o a c e t y l GoA A c e t y l- P + C O A a c e t o a c e t a t e t CoA The r e a c t i o n m echanism f o r th e o x id a t io n o f b u t y r y l G O A i s i d e n t i c a l t o t h a t fo u n d i n a n im a ls . The u n iq u e f e a t u r e s a r e th e a c t i v a t i o n o f a c e t a t e by p h o s p h o tr a n s - a c e t y l a s e and th e a c t i v a t i o n o f b u ty r a te and v i n y l a c e t a t e by a G O A t r a n s f e r a s e . K ornberg and P r ic e r (1 9 5 5 ) and B e in e r t and S t a n s ly (1 9 6 5 ) show ed t h a t a c t i v a t i o n o f f a t t y a c id s i n a n im a l s y s te m s ca n a l s o o c cu r th r o u g h a CoA t r a n s f e r a s e m ech an ism . A p a rt from t h e p r e v io u s l y d is c u s s e d w ork o f B a r k e r 's group w h ic h was c o n c e r n e d w it h th e m e ta b o lis m o f s h o r t c h a in f a t t y a c i d s , v e r y l i t t l e h a s b e e n p u b lis h e d on t h e 9 m echanism o f f a t t y a c id o x i d a t i o n by b a c t e r i a . S i H i k e r (1 9 5 0 ) and S i l l i k e r and R it t e n b e r g (1 9 5 1 a , 1 9 5 1 b , and 1 9 5 2 ) i n v e s t i g a t e d th e m echanism o f o x id a t io n o f in t e r m e d ia t e c h a in l e n g t h f a t t y a c id s by S e r r a t i a m a r c e s c e n s . T hey w orked w it h w h o le c e l l s e m p lo y in g th e te c h n iq u e o f s im u l ta n e o u s a d a p t a t io n and c e l l i n h i b i t o r s . T h ese i n v e s t i g a t i o n s in d ic a t e d t h a t th e m e ta b o lism o f t h e in te r m e d ia te c h a in le n g t h a c id s (C5 -C1 4 ) w ere m e d ia te d by a common enzym e s y s te m . I t was f u r t h e r shown t h a t th e a lp h a b e ta u n s a t u r a t e d , b e ta h y d r o x y , and b e t a k e to f a t t y a c id s w ere in t e r m e d ia t e s , or c l o s e l y r e l a t e d t o in t e r m e d ia t e s i n f a t t y a c id o x i d a t i o n . A ls o , i t was d e m o n str a te d t h a t lo w e r f a t t y a c id s a s su c h w ere n o t in t e r m e d ia t e s in t h e o x id a t io n o f h ig h e r f a t t y a c i d s . T h ese f i n d i n g s r u l e s o u t omega o x id a - I t i o n and b e t a o x i d a t i o n i n th e c l a s s i c a l s e n s e b u t c o u ld n o t d i s t i n g u i s h b e tw e e n t h e p o s s i b i l i t y o f m u lt ip le a l t e r n a t e o x id a t io n and b e ta o x i d a t i o n i n v o l v i n g a c t i v e i n t e r m e d ia t e s , I , THE PROBLEM As a f u r t h e r e x t e n s io n o f t h i s w ork i t w as d e c id e d t o i n v e s t i g a t e t h e o x id a t io n o f p h e n y l s u b s t i t u t e d f a t t y a c i d s . T h is a p p ro a ch was e n t i c i n g a t th e tim e s i n c e i t w as known t h a t b e n z o a te a n d p h e n y la c e t a t e a r e o x id iz e d 10 a d a p t iv e ly b y a v a r i e t y o f b a c t e r i a ( S t a n ie r , 1 9 5 0 ) and th u s c o n v e n ie n t m ark ers w o u ld be a v a i la b l e * I I t sh o u ld b e p o in t e d o u t t h a t t h e s e s t u d i e s w e re i n i t i a t e d I n 1950 b e f o r e t h e n a tu r e o f t h e a c t i v e a c e t a t e (L ynen e t a l , , 1 9 5 1 ) w as known and CoA in v o lv e m e n t i n f a t t y a c id o x id a t io n was d e m o n s tr a te d . The d a ta o b ta in e d i n s t u d y in g t h e p h e n y l s u b s t i t u t e d a c i d s , w h ic h c o n s t i t u t e s th e f i r s t p a r t o f t h i s , d i s s e r t a t i o n , le d t o e s s e n t i a l l y th e same c o n c lu s io n s a s th e p r e v io u s w ork by s i l l i k e r and R it t e n b e r g (1 9 5 1 a , 1 9 5 1 b , and 1 9 5 2 ) and f a i l e d t o r e v e a l th e d e t a i l e d m echanism o f f a t t y a c id o x id a t io n by t h i s o r g a n ism . C o n s e q u e n tly , a d i f f e r e n t a p p ro a ch was ta k e n i n w h ic h a tte m p ts w ere made t o o b ta in c e l l - f r e e sy ste m s from b a c t e r i a c a p a b le o f o x i d i z i n g f a t t y a c i d s . T h ese i n v e s t i g a t io n s w ere i n p r o g r e s s d u r in g th e p e r io d i n w h ich t h e d e t a i l s o f th e o x id a t io n by a n im a l sy s te m s w ere w orked o u t . The r e s u l t s o b ta in e d , w h ic h a r e r e p o r te d i n t h e se c o n d p a r t o f t h e d i s s e r t a t i o n , show i n g e n e r a l, a c l o s e s i m i l a r i t y b e tw e en b a c t e r i a l and a n im a l m ech a n ism s. CHAPTER II THE OXIDATION OP PHENYL SUBSTITUTED PATTY ACIDS BY RESTING CELLS OP VARIOUS BACTERIA I . INTRODUCTION D u rin g t h e c o u r s e o f i n v e s t i g a t i o n s on th e o x id a t io n o f f a t t y a c id s by S e r r a t i a m a r c e sc e n s ( A lp h in ) , d a ta w ere a c c u m u la ted w h ic h in d ic a t e d t h a t lo w e r f a t t y a c id s a s s u c h , w ere n o t d i r e c t in t e r m e d ia t e s i n th e o x id a t io n o f h ig h e r f a t t y a c id s ( S i l l i k e r and R it t e n b e r g , 1 9 5 1 a , 1 9 5 1 b , and 1 9 5 2 )# To expand t h e s e o b s e r v a t io n s i t w as d e c id e d t o i n v e s t i g a t e th e o x i d a t i o n o f p h e n y l s u b s t i t u t e d f a t t y a c id s by v a r io u s b a c t e r i a • T h is a p p ro a ch w as p a r t i c u l a r l y e n t i c i n g s i n c e i t i s known t h a t b e n z o a te and p h e n y la c e t a t e a r e o x i d i z e d .a d a p t i v e l y b y a v a r i e t y o f b a c t e r i a ( S t a n ie r , 1 9 5 0 ) and th u s c o n v e n ie n t m ark ers w ou ld be a v a i l a b l e , I I , M ATER lA IS AND METHODS The lo w e r p h e n y l f a t t y a c id s w ere a v a i l a b l e com m e r c i a l l y , The h ig h e r p h e n y l d e r i v a t i v e s , p h e n y lb u ty r a te th r o u g h p h e n y lla u r a t e w ere s y n t h e s iz e d by Mr, P . M, Magee and M r, W, R , B ras e n . D ep artm en t o f C h e m istr y , U n iv e r s i t y o f s o u th e r n C a l i f o r n i a , u s in g m o d i f i c a t i o n s o f e x i s t i n g p r o c e d u r e s , 12 F iv e o rg a n ism s w e re i n v e s t i g a t e d , S e r r a t i a m arce s - c e n s (A lp h in ) and Pseudom onas f l u o r e s c e n s P -6 w ere u s e d i n p r e v io u s f a t t y a c id s t u d i e s i n our la b o r a t o r y . Pseudom onas f l u o r e s c e n s A 3 ,1 6 and A 3 ,1 7 w ere o b ta in e d fro m D r , R , Y . S t a n i e r , U n iv e r s i t y o f C a l i f o r n i a , C u ltu r e S ,5 1 , p r o b a b ly a Pseudom onas f l u o r e s c e n s s t r a i n , was i s o l a t e d fro m s o i l by en r ic h m e n t c u lt u r e te c h n iq u e s u s in g p h e n y lb u ty r a t e a s th e s o l e ca rb o n s o u r c e . C e l l s w ere grow n i n a m in e r a l s a l t s s o l u t i o n (pH 7 ,0 ; c o n t a in in g 0 ,5 ^ K^HPO*, 0 , ^ KHgPO^, 0 , 1^ N % C l, 0 . 01^ MgSO* and e i t h e r th e u n s u b s t it u t e d f a t t y a c id ( 0 ,0 1 M ), th e p h e n y l s u b s t i t u t e d f a t t y a c id ( 0 ,0 0 1 M ), o r g lu c o s e (0,028M ) a s t h e s o l e c a r b o n s o u r c e . In c e r t a i n c a s e s w h ere h e a v y c e l l y i e l d s w ere d e s ir e d 0 ,1 p e r c e n t y e a s t e x t r a c t w as u s e d a s a su p p lem en t i n m ed ia c o n t a in in g th e p h e n y l su b s t i t u t e d a c id s a s th e c a r b o n s o u r c e . C e l ls w ere grow n i n 5 0 0 m l E r le n m e y er f l a s k s , 1 0 0 m l c u lt u r e medium p e r f l a s k , and a g i t a t e d i n a c o n s t a n t te m p e r a tu r e w a te r b a th a t 30 o r 37®C f o r 18 t o 7 2 h o u rs d e p e n d in g on th e s u b s t r a t e . A f t e r grow th c e l l s w ere h a r v e s t e d by c e n t r i f u g a t i o n , w ashed th r e e t im e s , and su sp en d ed t o a t u r b i d i t y o f a p p r o x im a te ly 3 0 0 (K lett-S u m m erso n p h o t o e l e c t r i c c o lo r im e t e r , 5 4 0 m illim ic r o n f i l t e r ) u s in g 0 ,0 5 M p h o sp h a te b u f f e r , pH 7 , 0 , S u b s t r a t e s w ere made up 13 i n a p p r o p r ia te c o n c e n t r a t io n s i n t h e same b u f f e r . O x id a tio n s t u d i e s w ere c a r r ie d o u t u s in g c o n v e n tio n a l W arburg te c h n iq u e s (U m b reit e t a l , , 1 9 4 9 ) , I I I , EXPERIMENTAL RESULTS O x id a tio n p a t t e r n s . T a b le I show s t h e o x id a t io n p a t t e r n s o f g lu c o s e grown c e l l s o f th e f i v e o r g a n ism s on th e p h e n y l s u b s t i t u t e d a c i d s . A l l o x id iz e d th e s u b s t r a t e s h a v in g a s i x ca rb o n c h a in l e n g t h or lo n g e r , t h e S e r r a t i a a f t e r an a p p r e c ia b le l a g , t h e o th e r s im m e d ia te ly . O xid a t i o n o f p h e n y lb u ty r a te or s h o r t e r c h a in s u b s t i t u t e d a c i d s , i f i t o c c u r r e d , in v o lv e d a la g i n a l l c a s e s . O nly th e s o i l i s o l a t e o x id iz e d a l l th e s u b s t r a t e s . The o th e r o rg a n ism s o x id iz e d e i t h e r b e n z o a te or p h e n y la c e t a t e b u t n o t b o th , and w ere in c a p a b le o f m e t a b o liz in g p h e n y lp r o p io n a te and p h e n y l- b u t y r a t e , The im m ed ia te o x i d a t i o n o f a f a t t y a c id s u b s t r a t e by g lu c o s e grow n c e l l s i s e v id e n é e t h a t t h e o rg a n ism c o n c e r n e d p o s s e s s e s c o n s t i t u t i v e enzym es f o r t h e d e g r a d a tio n o f t h e s u b s t r a t e . The o c c u r r e n c e o f a la g i n th e o x id a t io n o f a s u b s t r a t e ca n m ean t h a t a d a p t iv e enzym es a r e in v o lv e d a l th o u g h o th e r i n t e r p r e t a t i o n s a r e a l s o p o s s i b l e from su c h d a ta a l o n e . S in c e s t a n i e r (1 9 5 0 ) h a s shown t h a t th e o x i d a t io n o f b e n z o a te i s m e d ia te d by a d a p t iv e enzym es i n 14 TABIE I OXIDATION O P PHENYL SUBSTITUTED PATTY ACIDS BY FIVE BACTERIA O x id a tio n o f O rganism s < t > C 2 < t> C 3 # 4 $05 $ 05-^022 S , marc e s c e n s (A I p h in ) - L - - - L p , f l u o r e s c e n s A 3 ,1 6 - L - - - G P , f l u o r e s c e n s A 3 ,1 7 L - - € P , f l u o r e s c e n s P ,6 L - - - C C " S o i l i s o l a t e 8 .5 1 L L L L C G - = no o x id a t io n ; C = o x id iz e d c o n s t i t u t i v e l y ; L = è x id iz e d a f t e r l a g . R e a c t io n m ix tu r e c o n ta in e d 1 .0 m l g lu c o s e grown r e s t i n g c e l l s ( t u r b i d i t y o f 3 0 0 ; K l e t t f i l t e r N o, 5 4 ) and 1 pM. o f t h e in d ic a t e d s u b s t r a t e i n a t o t a l volum e o f 2 ,0 m l. I n c u b a t io n 3 7 ° C, a i r a tm o sp h e r e , 0,05M p h o sp h a te b u f f e r , pH 7 , 0 . 0 ,1 m l 2C^ KOH i n c e n t e r w e l l f o r COg a b s o r p t io n . 15 s t u d i e s w it h some o f t h e same o rg a n ism s u s e d i n t h i s i n v e s t i g a t i o n , and s i n c e t h e la g s i n o x i d a t i o n o b s e r v e d (T a b le I ) a r e e lim in a t e d i f t h e c e l l s a r e grow n on th e su b s t r a t e in v o lv e d ( s e e F ig u r e s 1 and 2 ) , i t i s h i g h ly p ro b a b le t h a t a d a p tiv e enzym es a r e in d e e d r e s p o n s ib l e f o r t h e la g s o b s e r v e d , c o n s e q u e n t ly , th e te c h n iq u e o f s im u lta n e o u s a d a p t a t io n ca n be a p p l ie d . A d a p ta tio n e x p e r im e n ts , C e l l s w ere a d a p ted t o v a r io u s compounds e i t h e r b y g ro w th on th e compound o r e l s e by a llo w in g g lu c o s e grow n r e s t i n g c e l l s t o o x i d i z e t h e compound i n b u f f e r s o l u t i o n . T h is l a t t e r t e c h n iq u e , term ed 'e x p o s u r e ', r e s u l t s i n a d a p tiv e enzym e fo r m a tio n , and t h e r e f o r e 'e x p o s e d ' c e l l s c a n b e u s e d i n s im u lta n e o u s a d a p t a t io n s t u d i e s . R e s t in g c e l l s u s p e n s io n s a d a p ted t o a p p r o p r ia t e compounds w ere t e s t e d f o r t h e i r o x id a t io n p a t t e r n and o x y g e n u p ta k e on p e r t i n e n t s u b s t r a t e s . F ig u r e s 1 and 2 show th e o x id a t io n p a t t e r n s o f fo u r t y p e s o f s ,5 1 c e l l s o x i d i z i n g p h e n y la c e t a t e and p h e n y l b u ty r a t e , C e l ls grow n on g lu c o s e or a d a p te d t o p h e n y l- c a p r y la t e o x id iz e d p h e n y la c e t a t e a f t e r a la g b u t show ed ^ l i t t l e o r n o o x y g en u p ta k e on p h e n y lb u ty r a t e . C e l l s grown on p h e n y lb u ty r a te o x id iz e d p h e n y la c e t a t e and p h e n y lb u ty r a te im m e d ia te ly , w h ereas p h e n y la c e t a t e grown c e l l s o x id iz e d th e hom ologou s compound im m e d ia te ly b u t f a i l e d t o o x i d i z e I C O I g M 120 100 8 0 60 4 0 20 6 0 L egen d: © = grow n; • = < |)C a grow n; A = g lu c o s e grow n; ^ = (|)Gs ex p o sed * R e a c t io n m ix tu r e c o n ta in e d 1 m l r e s t i n g c e l l s ( t u r b i d i t y o f 5 00; K l e t t - f i l t e r Ho* 5 4 ) , 1 yM p h e n y la c e t a t e i n a t o t a l volum e o f 2 * 0 ml# I n c u b a tio n 57® C, a i r a tm o s p h e r e , 0*05 M p h o sp h a te b u f f e r , pH 7 .0 # 0*1 m l 20^ KOH i n c e n t e r w e l l f o r COg a b s o r p tio n * C o r r e c te d f o r a u t o - r e s p i r a t i o n * FIGURE 1 OXIDATION OP PHENYlACilTATE BY SOIL ISOLàTS S*51 17 100 80 60 M O B 40 H 20- 120 180 60 MINUTES L egend: A = < t)C 8 • - (|)C4 e x p o s e d . grow n; R e a c t io n m ix tu r e c o n ta in e d b i d i t y 3 0 0 ; K l e t t - f i l t e r N o. 5 4 ) , volum e o f 2 .0 m l. grow n; o = (jïCg grow n; A = g lu c o s e 1 m l r e s t i n g c e l l s ( t u r - 1 p h e n y lb u ty r a te i n a t o t a l.v o lu m e o f 2 .0 m l. I n c u b a tio n 37® C, a i r a tm o sp h e r e , 0 .0 6 M p h o sp h a te b u f f e r , pH 7 , 0 . 0 .1 m l 20^ KOH i n c e n te r w e l l f o r COg a b s o r p t io n , c o r r e c t e d f o r a u t o r e s p i r a t i o n . FIGURE 2 OXIDATION OF PHENYLBUTYRATE BY SOIL ISOLATE S . 51 p h e n y lb u t y r a t e . From t h e s e r e s u l t s i t i s a p p a r e n t t h a t a d a p t a t io n t o p h e n y lc a p r y la te f a i l e d t o a d a p t thfe c e l l s t o p h e n y lb u ty r a te or p h e n y la c e t a t e o x i d a t i o n . On t h e o th e r h an d , a d a p t a t io n t o p h e n y lb u ty r a te d id a d a p t th e c e l l s t o p h e n y la c e t a t e o x i d a t i o n . D a ta on th e t o t a l o x y g en co n sumed w it h t h e s e compounds a s s u b s t r a t e s (T a b le I I ) show t h a t th e o x id a t io n o f p h e n y lc a p r y la te m ust have go n e beyon d t h e b e n z o a te s t a g e and r u p tu r e o f th e r in g ( 1 2 ,5 jiM Og t o b e ta k e t o a d ip ic a c i d ) . I t i s p r o b a b le t h a t a l l t h r e e su b s t r a t e s a r e c o m p le te ly o x id iz e d and t h a t t h e d i f f e r e n c e s b e tw e en th e o b se r v e d and c a l c u l a t e d ox y g en u p ta k e s a r e due t o o x i d a t i v e a s s i m i l a t i o n . S in c e p h e n y lc a p r y la t e i s o x id iz e d beyond t h e o x i d a t io n s t a g e s o f p h e n y lb u ty r a te and p h e n y la c e t a t e w ith o u t a d a p tin g t o them , t h e s e com pounds, a s s u c h , c a n n o t b e in t e r m e d ia t e s i n t h e o x id a t io n o f p h e n y lc a p r y la t e . The d a ta s u g g e s t t h a t th e a c t i v e form s o f t h e s e com pounds a r e th e d i r e c t in t e r m e d ia t e s o f p h e n y lc a p r y la t e o x id a t io n and t h a t a d a p t a t io n t o t h i s compound d o e s n o t in d u c e t h e p r o d u c t io n o f enzym es n e c e s s a r y f o r th e a c t i v a t i o n o f p h e n y l b u ty r a t e and p h e n y la c e t a t e , A s i m i la r i n t e r p r e t a t i o n h a s b een p r e s e n t e d by S tad tm an and B ark er (1 9 4 9 b ) i n t h e s tu d y o f a l i p h a t i c a c id o x i d a t i o n . The a b i l i t y o f p h e n y lb u ty r a te grown c e l l s t o o x i d i z e 18 I I 19 TABLE I I OXYGEN UPTAKE O N VARIOUS PHENYL SUBSTITUTED PATTY ACIDS BY THE SOIL ISOLATE S . 61 S u b s t r a t e M oles o f o x y g en u s e d p e r m ole o f s u b s t r a t e O bserved C a lc u la t e d t o (j)C 4 C a te c h o l C o m p le tio n 6 ,2 - — 2 ,5 9 < |)C 4 9 ,5 3 5 ,5 12 4)08 1 2 ,0 6 9 1 1 ,5 18 R e a c tio n m ix tu r e c o n ta in e d 1 m l r e s t i n g c e l l s ( t u r b i d i t y o f 3 0 0 ; K le t t f i l t e r N o ,.5 4 ) and 1 jiM o f t h e i n d ic a t e d s u b s t r a t e i n a t o t a l volum e o f 2 ,0 m l. I n c u b a t io n 37® G, a i r a t m o s p h e r e ,0 ,0 5 M p h o sp h a te b u f f e r , pH 7 , 0 , 0 ,1 m l 20^ KOH i n c e n te r w e l l f o r COg a b s o r p t io n . C or r e c t e d f o r a u t o r e s p i r a t i o n , D a ta p r e s e n te d a r e a v e r a g e s o f s e v e r a l d e t e r m in a t io n s . -— ---------------------- ---- — — — —— --------------------------- — I 20 ! p h e n y la c e t a t e w ith o u t a l a g w ou ld i n d i c a t e t h a t t h i s l a t t e r compound c o u ld h e a n in t e r m e d ia t e in th e o x id a t io n o f p h en y lb u t y r a t e • I n a n o th e r ty p e o f a d a p t a t io n e x p e r im e n t, c a p r a te grow n c e l l s o f A 3 ,1 7 w ere a llo w e d t o o x i d i z e b e n z o a te and p h e n y lh e p ty l a t e i n a W arburg f l a s k (F ig u r e 3 ) , When o x i d a t io n o f t h e s e compounds c e a s e d , a se c o n d p o r t io n o f b e n z o a te was t ip p e d i n t o e a c h f l a s k . C e l l s p r e v io u s ly o x i d i z in g b e n z o a te show ed an im m ed iate o x y g en u p ta k e when g iv e n th e same s u b s t r a t e i n d ic a t in g t h a t a d a p ta tio n had o c c u r r e d , C e l l s p r e v io u s ly o x i d i z i n g p h e n y I h e p t y la te had th e same l a g i n b e n z o a te o x id a t io n a s d id t h e o r i g i n a l c e l l s , sh ow in g t h a t a d a p t a t io n t o th e lo w er compound had n o t o c c u r r e d , S im ila r r e s u l t s w ere o b ta in e d w it h p ,6 and A 3 ,1 6 i n t h a t o x id a t io n o f a h ig h e r odd or e v e n c h a in p h e n y l f a t t y a c id d id n o t a d a p t t o t h e o x i d a t i o n o f b e n z o a te or p h e n y la c e t a t e r e s p e c t i v e l y . The s i g n i f i c a n c e o f th e f a i l u r e o f t h e s e t h r e e o r g a n ism s t o a d a p t t o t h e a p p r o p r ia te lo w e r s u b s t r a t e i s som ewhat d i f f e r e n t th a n f o r s * 5 1 . An e x a m in a tio n o f t h e t o t a l o x y g en consum ed (T a b le I I I ) show s t h a t o x id a t io n was in c o m p le t e . I n t h e c a s e o f P,6 and A 3 ,1 7 , o x id a t io n ap p a r e n t ly d o e s n o t p r o c e e d beyond th e b lo c k a t p h e n y l- p r o p io n a te and a d a p t a t io n to b e n z o a te ca n n o t b e e x p e c te d . 2V % g o C O I 6 y 100 8 0 6 0 4 0 20 0 60 120 180 240 500 360 MINUTES L egend: • = 0 .6 p h e n y Ih e p ty l a t e ; © = 0 .5 )iM b e n z o a te ; -►= 0 .5 pM b e n z o a te a d d ed . R e a c t io n m ix tu r e c o n ta in e d 1 m l r e s t i n g c e l l s ( t u r b i d i t y o f 3 0 0 ; K l e t t - f i l t e r N o. 5 4 ) and th e I n d ic a t e d s u b s t r a t e i n a t o t a l volum e o f 3 . 0 m l. In cu b a t i o n 37® Cf a i r a tm o sp h e r e , 0 .0 5 M p h o sp h a te b u f f e r , pH 7 . 0 . 0 .1 m l 20^ KOH i n c e n t e r w e l l f o r COg a b s o r p t io n . C o r r e c te d f o r a u t o r e s p i r a t i o n . FIGURE 3 OXIDATION O F PEENYLTBPTYIATE AND BENZOATE BY P . FLUORESCENS A 3 .1 7 22 TABIE III OXYGEN UPTAKE O N VARIOUS PBENYL SUBSTITUTED PATTY ACIDS BY THREE PSEUDOMONADS AND S . MARGES CENS (ALPHIN) O rgan ism Sub s t r a t e M oles o f o x y g en u s e d p e r m ole o f s u b s t r a t e Obs e r v e d C a lc u la te d t o # 4 # 3 # 2 # 1 C o m p le tio n P . 6 4 . 0 - - - - 7 .5 1 .9 - 3 6 1 3 .5 # 7 3 .5 - 6 - 9 1 6 .5 A 3 .1 7 4ici 3 .9 - - 7 .5 4 .6 - 6 - 9 1 6 .5 A 3 .1 6 (jJC g 5 .5 - - - - 9 .0 <|lG g 5 . 2 3 - 6 - 1 5 .0 A lp h in 1 1 .5 9 - 12 - 2 1 .0 R e a c tio n m ix tu r e c o n ta in e d 1 ml r e s t i n g c e l l s ( t u r b i d i t y o f 3 0 0 ; K l e t t - f i l t e r N o. 5 4 ) and 1 pM o f th e i n d ic a t e d s u b s t r a t e i n a t o t a l volum e o f 2 .0 m l. I n c u b a tio n 37® Of a i r a tm o sp h e re , 0 .0 5 M p h o sp h a te b u f f e r , pH 7 . 0 . 0 .1 m l 20^ KOH i n c e n te r w e l l f o r COg a b s o r p t io n . C or r e c t e d f o r a u t o r e s p i r a t i o n . D a ta p r e s e n t e d a r e a v e r a g e s o f s e v e r a l d e t e r m in a t io n s . 23 In th e c a s e o f A 3 .1 6 , th e o x id a t io n p r o c e e d s beyond t h e ap p a r e n t b lo c k a t p h e n y lb u ty r a te b u t n o t to c o m p le tio n . W hether th e o x id a t io n g o e s b eyon d t h e p h e n y la c e t a t e s t a g e i s u n c e r t a in b e c a u se th e amount o f o x i d a t i v e a s s i m i l a t i o n o c c u r r in g d u r in g t h e o x id a t io n o f t h e s i d e c h a in i s u n known. F ig u r e 4 show s th e o x id a t io n p a t t e r n o f g l u c o s e and c a p r a te grow n s . marc e s c e n s ( A lp h in ) . P h e n y la c e t a t e o x i d a t io n i s a d a p tiv e by b o th t y p e s o f c e l l s . C a p ra te grown c e l l s , h o w ev e r, a r e a d a p ted t o th e o x id a t io n o f p h e n y l- c a p r a te sh ow in g t h a t th e enzym es in v o lv e d i n th e o x id a t io n o f th e a l i p h a t i c a c i d a l s o f u n c t i o n i n th e o x id a t io n o f t h e omega p h e n y l s u b s t i t u t e d com pound. A lth o u g h c a p r a te grow n c e l l s a r e n o t a d a p te d t o p h e n y la c e t a t e o x id a t io n , t h e sam e c e l l s a f t e r o x id a t io n o f p h e n y lc a p r a te a r e s o a d a p t e d .. T h is i s s e e n from c u r v e A F i g u r e 4 , sh ow in g im m ed ia te o x y g en u p ta k e when p h e n y l a c e t a t e i s tip p e d i n a t t h e end o f p h e n y lc a p r a te o x i d a t i o n . An e x a m in a tio n o f t h e same c u r v e show s a marked b rea k i n th e o x id a t io n r a t e a t 120 m in u te s . The r a t e and t o t a l o x y g en u p ta k e beyon d t h i s p o in t a r e a b o u t th e same a s t h a t u l t i m a t e l y o b ta in e d d u r in g p h e n y la c e t a t e o x i d a t i o n by c a p r a te grow n c e l l s . F or t h i s o rg a n ism i t a p p e a r s t h a t t h e o x id a t io n o f e v e n c h a in p h e n y l f a t t y a c id s p r o c e e d s w ith P H ® 2 4 P 0 ^ P © U Pi <1 44 44 0 b O ta p ^ ,P © © 3 P i TJ C Q 45 n © © 0 P 0 44 0 44 • *d 0 0 < D 0 A < D rH O 4 ) -P \ U II ^ O S ^ I Q Q 0 'O O # e < D a «, ^ 4» v4 o . P i C d P • « k o S a s i g " ^ r H U P i 0 v4 4 a & O S o n o o § H O 4 » I 4>0 • t ïO 0 3 4 > C O > © 4 a 4 h 44 O S o p 0 3 O S 44 O H © H t4 M P i O 8 44 ( D v S G S O S O O r4 M n i" i'ss 2 - 4 - a a " b O ••» P © 44 • © 44 P H 44 p 0 a P f i r4 0 U P i 0 P i P • © « P 0 © U f c O 0 H > P . ^ 44 q_i II P © M 0 I — I r4 % k I o © r4 y O ^ H S © ° p , © § O P P .. ^ OH m r > O b O . 1 0Î a H 0 0 1 • H 44 0 % cS H M Pi b O © P CP «P o 44 PiH P i 1 m fi © © U Pj^H o 5 I1 1 53 I « a j I i C P col I o P 4 I â Pi § M E H i MICROLITHîS OXYGEN p h e n y la c e t a t e or a s i m i l a r compound te m p o r a r ily a c c u m u la tin g a s an end p r o d u c t o f t h e s i d e c h a in o x i d a t i o n . T h is p ro d u c t i s th e n o x id iz e d th r o u g h a path w ay w h ich i s t h e sam e a s t h a t f o r p h e n y la c e t a t e . F i n a l l y I t sh o u ld be p o in t e d o u t t h a t a t o t a l o f b e tw een 11 and 12 M o f oxygen a r e consum ed p e r m o le o f p h e n y l c a p r a te (T a b le I I I ) and t h a t o n ly 9 a r e r e q u ir e d t o b r in g t h i s compound t o t h e p h e n y lb u ty r a te s t a g e w h ich i s n o t o x id iz e d a t a l l b y 8 . marc e s c e n s ( A lp h in ) . IV . DISCUSSION F o r a l l b u t one o f th e o r g a n ism s t e s t e d t h e r e i s c o n c lu s iv e e v id e n c e t o show t h a t lo w e r p h e n y l f a t t y a c id s a s su c h a r e n o t d i r e c t in t e r m e d ia t e s i n th e o x id a t io n o f th e h i g h e r - h o m o lo g u e s. S . 51 o x i d i z e s , p h e n y lc a p r y la te t o c o m p le tio n w ith o u t a d a p tin g t o p h e n y lb u ty r a te or p h e n y l a c e t a t e . S . m arc e s c e n s , o x i d i z e s p h e n y lc a p r a te p a s t t h e p h e n y lb u ty r a te s t a g e w h ic h i t i s in c a p a b le o f o x i d i z i n g . S i m i l a r l y , A 3 .1 7 and A 3 .1 6 o x id iz e lo n g e r c h a in p h e n y l f a t t y a c id s p a s t p h e n y lv a le r a t e and p h e n y lb u ty r a te r e s p e c t i v e l y , w h ic h l i k e w i s e a r e n o t d e g r a d e d . On th e o th e r h an d , a d a p t a t io n o f S . 5 1 t o p h e n y l b u t y r a t e s im u lt a n e o u s ly a d a p ts t o th e o x id a t io n o f p h e n y l a c e t a t e ; o x i d a t i o n o f p h e n y lc a p r a te b y s . m a rc esce n s 26 s im u lt a n e o u s ly a d a p ts t o th e o x id a t io n o f p h e n y la c e t a t e ; o x id a t io n o f h ig h e r e v e n and odd c h a in f a t t y a c id s by S . m a r c e s c e n s , P .6 , and A 3 .1 7 a p p a r e n tly r e s u l t s i n t h e a c c u m u la tio n , tem p o ra ry o r p erm a n en t, o f p h e n y Ip r o p io n a t e o r p h e n y la c e t a t e . A l l t h e s e d a ta im p ly b e ta o x id a t io n i s o c c u r r in g . The ab ove d a ta a p p ea r c o n t r a d ic t o r y , h o w ev er, m ost o f t h e r e s u l t s o b ta in e d c a n b e r e c o n c il e d on t h e b a s i s o f th e f o llo w in g c o n s i d e r a t i o n s . W ith t h e p h e n y l r a d i c a l on th e t e r m in a l c a rb o n i t i s a p p a r e n t t h a t a f a t t y a c id o x i d i z i n g m echanism i s n o t s u f f i c i e n t f o r a c o m p le te o x id a t io n o f t h e s u b s t r a t e . From t h e i n i t i a l work o f Knoop (1 9 0 4 ) i n w h ic h b e n z o a te o r p h e n y la c e t a t e a c c u m u la ted i n a n im a l f e e d in g e x p e r im e n ts , one can c o n c lu d e t h a t i n a n im a ls a t l e a s t , th e f a t t y a c id o x i d i z i n g m echanism ca n h a n d le a l l b u t b e n z o a te and p h e n y la c e t a t e . T h is may a l s o .b e t r u e f o r t h e b a c t e r i a l f a t t y a c id o x i d i z i n g s y s te m , e v en th o u g h fo u r o f th e f i v e o r g a n ism s u se d c a n n o t m e t a b o liz e p h e n y lb u ty r a te or p h e n y lp r o p io n a te a s. s u c h . S in ce , s . marc e s c e n s h a s b e e n shown in c a p a b le o f u s in g p r o p io n a te and b u t y r a t e ( S i l l i k e r , 1 9 6 0 ) and P-6 in c a p a b le o f u s in g b u t y r a t e t h i s i n a b i l i t y i s a p p a r e n tly in d e p e n d e n t o f th e p h e n y l s u b s t i t u t i o n . R e c e n t e x p e r im e n ta l d a ta from a n im a l sy s te m s show t h a t th e o x id a t io n o f a l i p h a t i c a c id s o c c u r s by a 27 1 p r e lim in a r y a c t i v a t i o n r e q u ir in g ATP and CoA f o llo w e d by b e t a o x id a t io n o f t h e a c t i v a t e d s u b s t r a t e (M a h ler, 1 9 5 3 b ). S im ila r f i n d in g s w ere r e p o r te d f o r th e b a c te r iu m C lo s tr id iu m k lu y v e r l w it h r e s p e c t t o th e lo w e r a l i p h a t i c a c i d s , p a r t i c u l a r l y b u ty r a te (S ta d tm a n e t a l . , 1 9 5 1 ) . As i s shown i n l a t e r s e c t i o n s o f t h i s t h e s i s , a c t i v a t i o n o f c a p r a te i s a n e c e s s a r y p r e r e q u i s i t e t o i t s o x id a t io n by a t l e a s t tw o o f t h e e x p e r im e n ta l o r g a n ism s, S . m arce s c e n s and p .6 . S in c e i t h a s b een in d ic a t e d by s im u lta n e o u s a d a p ta t i o n e x p e r im e n ts w it h S . m a r c e sc e n s t h a t th e sam e enzym es f u n c t i o n i n th e o x id a t io n o f a l i p h a t i c and p h e n y l su b s t i t u t e d f a t t y a c i d s , i t i s p r o b a b le t h a t f o l l o w i n g an i n i t i a l ATP and CoA a c t i v a t i o n o f a p h e n y l s u b s t i t u t e d f a t t y a c i d , b e ta o x i d a t i o n o f th e a c t i v a t e d compound o c c u r s . A l l o f th e o r g a n ism s u a ed can m e t a b o liz e e i t h e r b e n z o a t e .o r p h e n y la c e t a t e or b o t h . The enzym e sy s te m s i n v o lv e d h e r e m ust b e d i f f e r e n t from t h e f a t t y a c id o x i d i z i n g s y s te m , and f o r t h e P seudom onads, a t l e a s t , b e n z o a te and p h e n y la c e t a t e w ould be o x id iz e d th r o u g h th e a r o m a tic p a th w ays d e s c r ib e d by o th e r s ( s t a n i e r , 1 9 5 0 and E v a n s, 1 9 4 7 ) . F or c o m p le te o x i d a t i o n o f a p h e n y l f a t t y a c id t h e f o l lo w in g s t e p s c o u ld th e n b e p o s t u la t e d : ( 1 ) a c t i v a t i o n ; ( 2 ) b e ta o x id a t io n t o b e n z o y l CoA or p h e n y la c e t y l CoA; and ( 3 ) d é a c y la t i o n and t r a n s f e r t o t h e r in g o x i d i z i n g sy ste m 28 and c o m p le te d e g r a d a tio n th r o u g h c a t e c h o l and b e ta k e t o a d ip ic a c id , or some o th e r p ath w ay, t o ca rb o n d i o x i d e and w a t e r . T h is m echanism w ou ld a c c o u n t f o r th e d a ta o b ta in e d w it h a l l o f th e o r g a n ism s e x c e p t 8 . 5 1 . The P seudom onads, A 3 .1 6 , A 3 .1 7 , and P . 6 c a r r y o u t t h e a c t i v a t i o n and b e ta o x i d a t io n , b u t t h e l a t t e r m echanism i s b lo c k e d b e f o r e th e t r a n s f e r p o in t (T a b le I I I ) . W ith s . m a r c e sc e n s (A lp h in ) a l l t h r e e s t e p s f u n c t i o n w it h th e tem p o ra ry a c c u m u la tio n o f an in t e r m e d ia t e , p r o b a b ly p h e n y la c e t a t e , a llo w in g a d a p ta t i o n t o o c c u r . W ith c a p r a te grow n c e l l s o f t h i s o rg a n ism th e r a t e o f t h e r in g o x i d a t i v e pathw ay i s s lo w e r th a n th e b e t a o x i d a t i o n m echanism a s e v id e n c e d b y a r a t e ch an ge d u r in g p h e n y Ic a p r a te o x id a t io n (F ig u r e 4 , c u r v e A ) . To e x p la in t lie d a ta o b ta in e d w it h 8 .5 1 , i t i s n e c e s s a r y t o p o s t u l a t e tw o o x id a t iv e m ech a n ism s, one i n v o lv in g th e o x id a t io n o f a c t i v a t e d com pounds, t h e o th e r t h e o x i d a t i o n o f th e p h e n y l f a t t y a c id s a s s u c h . The i n a b i l i t y o f p h e n y lc a p r y la t e grown c e l l s t o a d a p t t o p h e n y lb u ty r a te and p h e n y la c e t a t e w o u l d i n d i c a t e th e n o n -fo r m a tio n o f t h e s e compounds p e r s e d u r in g th e o x id a t io n o f p h e n y lc a p r y ly l CoA. In o t h e r w o r d s, t h e r e w ou ld be no d é a c y la t io n o f p h e n y la c e t y l CoA or p h e n y I b u ty r y l coA d u r in g o x i d a t i o n . The a b i l i t y o f p h e n y lb u ty r a te grown c e l l s t o a d a p t t o 29 p h e n y la c e t a t e o x id a t io n can b e e x p la in e d i n tw o w a y s . E i t h e r p h e n y lb u ty r a te i s o x id iz e d d i r e c t l y w ith p h e n y l a c e t a t e a s an in t e r m e d ia t e or e l s e b o th a r e b r o u g h t i n t o an o x id a t iv e p ath w ay b y t h e same a c t i v a t i n g en zym e. The l a t t e r p o s s i b i l i t y i s e lim in a t e d s i n c e p h e n y la c e t a t e o x id a t io n d o e s n o t a d a p t t h i s o rg a n ism t o t h e o x id a t io n o f p h e n y l- \ b u t y r a t e . A sc h e m a tic r e p r e s e n t a t io n o f p h e n y l f a t t y a c id o x id a t io n by a l l o f th e e x p e r im e n ta l o r g a n ism s i s p r e s e n te d i n F ig u r e 5 . V . SUM M A R Y I t was shown t h a t a v a r i e t y o f b a c t e r i a a r e a b le t o p a r t i a l l y or c o m p le t e ly o x i d i z e c e r t a i n p h e n y l s u b s t i t u t e d f a t t y a c i d s . On th e b a s i s o f a d a p t a t io n p a t t e r n s and o x y g en u p ta k e d a t a , i t h a s b e e n d e m o n str a te d ( 1 ) t h a t s h o r t e r p h e n y l f a t t y a c id s a s su c h a r e n o t d i r e c t i n t e r m e d ia te s i n t h e o x id a t io n o f lo n g e r p h e n y l s u b s t it u t e d f a t t y a c id s and ( 2 ) t h a t n e v e r t h e l e s s a b e t a o x id a t io n o f t h e lo n g e r c h a in compounds o c c u r s . The d a ta i n d i c a t e t h a t th e o x id a t io n o f lo n g c h a in p h e n y l f a t t y a c id s i n v o l v e s an a c t i v a t i o n by th e same enzym e( s ) in v o lv e d i n t h e a c t i v a t i o n o f u n s u b s t it u t e d a l i p h a t i c a c i d s , f o llo w e d b y a b e ta o x i d a t i o n o f th e 30 - # 1 0 ATP, CoA < t l P a t t y a c id CoA # 3 S . 51 # 1 -» — Odd E ven A 3 .1 7 ,P .6 b lo c k e d A 3 .1 6 b lo c k e d S .5 1 S . m a r c e sc e n s 4 B enzoyl-C oA ^ A c e t y l CoA- G O 8 .6 1 8 .6 1 , 8 . mar- c e s c e n s , A S.16 COe + HaO FIGURE 6 SCHEMâTIG DIAGRAM - OP PHENYL FATTY ACID OXIDATION BY FIVE. BACTERIA 51 a c t i v a t e d compounds w h ic h i n some o rg a n ism s p r o c e e d s t o th e b e n z o a te o r p h e n y la c e t a t e l e v e l * The o x id a t io n o f th e lo w e r p h e n y l f a t t y a c id s (p h e n y lb u ty r a te t o b e n z o a te ) i n fo u r o f t h e o r g a n ism s t e s t e d a p p a r e n tly p r o c e e d s v i a th e a r o m a tic schem e d e sc rib ed , b y S t a n ie r (1 9 5 0 ) and E vans ( 1 9 4 7 ) . H ow ever, t h e d a ta s u g g e s t t h a t an a l t e r n a t e p ath w ay p r o c e e d in g d i r e c t l y from t h e a c t i v a t e d p h e n y la c e t a t e and b e n z o a te a l s o e x i s t s i n th e s o i l i s o l a t e 8 .5 1 * CHAPTER III THE OXIDATION OP ALIPHATIC PATTY ACIDS BY CELL-PREE SYSTEMS PROM SERRATlA MARCESCENS (ALPHIN) I . INTRODUCTION P r e v io u s s t u d i e s w it h r e s t i n g c e l l s o f S* m a r c e sc e n s (A lp h in ) e m p lo y in g th e te c h n iq u e o f s im u lta n e o u s a d a p t a t io n and i n h i b i t i o n s t u d i e s e lim in a t e d v a r io u s p o s s i b i l i t i e s b u t d id n o t e s t a b l i s h th e o x i d a t i v e p ath w ay i n a l i p h a t i c a c id o x id a t io n ( S i l l i k e r and R it t e n b e r g , 1 9 5 1 a , 1 9 5 1 b , and 1 9 5 2 ), B e fo r e th e o x i d a t i v e p ath w ay o f f a t t y a c id o x id a t io n by a n im a ls c o u ld be e lu c id a t e d i t w as n e c e s s a r y t o d e v e lo p te c h n iq u e s f o r o b t a in in g a c t i v e s o l u b l e enzym e s y s t e m s . U s in g s o l u b l e sy s te m s from a n im a l t i s s u e s i t was show n t h a t t h e i n i t i a l s t e p i n t h e a e r o b ic m e ta b o lism o f f a t t y a c id s w as an ATP and CoA a c t i v a t i o n o f t h e a c id s u b s t r a t e t o an a c t i v a t e d d e r i v a t i v e ( a c y l Coenzyme A ) w h ic h t h e n u n d erw en t b e t a o x i d a t i o n . A s i m i la r a c t i v a t i o n and b e ta o x id a t io n w ere i n d ic a t e d i n th e s t u d i e s w it h th e p h e n y l f a t t y a c i d s , c o n s e q u e n t ly f a t t y a c id o x id a t io n by c e l l - f r e e e x t r a c t s o f b a c t e r i a was in v e s t i g a t e d # I n i t i a l e x p e r im e n ts i n d ic a t e d th e a b i l i t y o f c e ll-^ f r e e e x t r a c t s o f S . m a r c e sc e n s t o o x i d i z e th e p h e n y l s u b s t i t u t e d f a t t y a c i d s . H ow ever, t h e a l i p h a t i c f a t t y 33 a c id s w ere u t i l i z e d s i n c e th e y w ere more s o l u b l e i n aq u eo u s sy s te m s and p o s t u la t e d in t e r m e d ia t e s i n t h e i n i t i a l s t a g e s o f o x id a t io n w ere a v a i l a b l e . The a n a l y s i s o f o x id a t io n p r o d u c ts w ou ld a l s o be s i m p l i f i e d s i n c e t e c h n iq u e s w ere a v a i l a b l e from s t u d i e s w it h c e l l - f r e e a n im a l s y s t e m s . I I . METHODS P r e p a r a tio n o f c e l l - f r e e e x t r a c t s . S . m a r c e sc e n s (A lp h in ) c e l l s w ere grow n a s p r e v io u s l y d e s c r ib e d . A t th e end o f a 7 2 hou r g r o w th p e r io d t h e c e l l s w ere s e p a r a te d by c e n t r i f u g a t i o n i n th e c o ld (4®C t 1®€) a t 2 0 ,0 0 0 x g . The com pact m ass o f b a c t e r i a was t h e n r e su sp e n d e d i n d i s t i l l e d w a te r , th o r o u g h ly a g i t a t e d , and r e c e n t r i f u g e d . T h is p r o c e s s was r e p e a te d u n t i l th e c e l l s had b e e n w ashed w it h 10 v o lu m es p e r c e n t o f d i s t i l l e d w a te r i n r e s p e c t t o t h e o r i g i n a l m edium . A t th e end o f th e w a sh in g p e r io d , t h e c e n t r i f u g e tu b e s c o n t a in in g t h e p a ck ed m ass o f b a c t e r i a w ere d r a in e d by i n v e r t i n g on a b s o r b e n t p a p e r t o w e lin g . To a g iv e n w e ig h t o f m o is t c e l l s c o n ta in e d w i t h i n an i c e c o ld m o rta r w as added 2 .0 t o 2 .5 tim e s t h e w e ig h t o f f i n e pow dered g l a s s^ o r . l e v i g a t e d a lu m in a . The m ix tu r e 1 P y rex g l a s s pow der u s u a l l y em p loyed f o r m aking th e f i n e g r a d e s i n t e r e d g l a s s f i l t e r s w as u s e d . T h is m a t e r ia l was g e n e r o u s ly p r o v id e d by t h e c o r n in g G la s s Company th r o u g h th e c o u r t e s y o f Mr. R . A . P e t t y and Mr. S . L . J o l l y , 54 was th e n ground w it h maximum hand p r e s s u r e u n t i l t h e co n s i s t e n c y ch an ged from a d r y , pow dery s t a t e t o a m o is t , ta c k y on e (a p p ro x im a te tim e , 4 m in u t e s ) . A t th e end o f th e g r in d in g p e r io d t h e cream y p a s t e was t r a n s f e r r e d t o c o ld c e n t r i f u g e t u b e s , e x t r a c t e d w it h 5 m l c o ld p h o sp h a te b u f f e r fpH 7 . 0 ) p e r gram w et c e l l w e ig h t , and c e n t r if u g e d a t 2 0 ,0 0 0 X g f o r 50 m in u te s in t h e c o ld (4®c t 1®G). The r e s u l t i n g s u p e r n a ta n t was th e n u s e d im m e d ia te ly . M anom etric d e t e r m in a t io n . O xygen u p ta k e w as d e te r m in e d by c o n v e n t io n a l W arburg te c h n iq u e s (U m b reit e t a l . , 1 9 4 9 ) . P ro d u c t a n a l y s i s . I d e n t i f i c a t i o n o f th e en d p r o d u cts o f t h e r e a c t i o n was a c c o m p lish e d by ch r o m a to g r a p h in g t h e i r h yd roxam ic d e r i v a t i v e s . One m i l l i l i t e r o f th e r e a c t i o n m ix tu r e t o be t e s t e d was ad d ed t o 5 v olu m es o f c o ld a b s o lu t e e t h a n o l. The r e s u l t i n g p r e c i p i t a t e w as c o l l e c t e d b y c e n t r i f u g a t i o n i n th e c o ld (4®C) and d is c a r d e d . The s u p e r n a ta n t f l u i d was th e n a c i d i f i e d t o pH 2 and e x t r a c t e d w it h 10 t o 20 s m a ll p o r t io n s o f e t h e r . F iv e m i l l i l i t e r s o f e th a n o l w ere added and th e e t h e r rem oved a t 60® G on a w a te r b a t h . The r e s u l t i n g a l c o h o l i c r e s id u e was e s t e r i f i e d b y r e f l u x i n g th e e t h a n o l r e s id u e and 1 d rop o f c o n c e n tr a te d s u l f u r i c a c id u n d er a c o ld f i n g e r tu b e f o r 30 m in u te s . The 35 I h yd roxam ic a c i d s w ere form ed from th e r e s u l t i n g e s t e r s by I t h e a d d it io n o f h y d ro x y la m in e i n a c c o r d a n c e w it h th e p r o - c e d u r e o f S tad tm an and B ark er ( 1 9 5 0 ) . H ydroxam ic sta n d a r d s w ere p r e p a r ed from t h e i r c o r r e s p o n d in g e t h y l e s t e r s a f t e r t h e m ethod o f K orn berg and P r ic e r ( 1 9 5 1 ) . The h yd roxam ic a c id s w ere ch rom atograp h ed u n id im e n - s i o n a l l y a t room te m p e r a tu r e u t i l i z i n g Whatman N o. 1 f i l t e r p a p er s h e e t s swamped w it h a c e t i c a c i d . B u ta n o l-w a te r - a c e t i c a c id ( 4 : 5 : 1 ) was u s e d a s t h e d e v e lo p e r in a c c o r d a n c e w it h th e m ethod o f Lugg and O v e r e ll (1 9 4 8 ) w it h t h e e x c e p t io n t h a t a s c e n d in g d e v e lo p m en t w as e m p lo y e d . A f t e r d r y in g , s p o t s w ere d e v e lo p e d w it h th e f e r r i c c h lo r id e d e v e lo p e r o f S tad tm an and B a rk er ( I 9 6 0 ) . I l l . EXPERIMENTAL RESULTS C o fa c to r r e q u ir e m e n ts . The e f f e c t o f v a r io u s c o f a c t o r s on th e a b i l i t y o f cru d e c e l l - f r e e e x t r a c t s t o o x i d i z e c a p r ic and c a p r y li c a c id s w as d e te r m in e d . In t h e f i r s t e x p e r im e n t r e a c t i o n v e s s e l s w ere s e t up i n tw o s e r i e s , S e r i e s one c o n ta in e d e i t h e r c a p r ic or c a p r y lic a c id s a s s u b s t r a t e p lu s t h e cru d e enzym e p r e p a r a tio n w ith o u t f u r t h e r a d d i t i o n s . S e r i e s tw o c o n t a in e d th e a c id s u b s t r a t e s , t h e cru d e enzym e p r e p a r a tio n and th e f o l l o w i n g c o f a c t o r s : ATP, CoA, CSH, Mg, C ytochrom e c , DPN, s u c c i n i c a c id , and r i b o - 36 f l a v in e - 5 - p h o s p h a t e . Prom th e r e s u l t s r e c o r d e d i n T a b le IV i t ca n be s e e n t h a t t h e c e l l - f r e e e e x t r a c t s w ere in c a p a b le o f o x i d i z i n g c a p r ic or c a p r y li c a c id s u n le s s t h e su p p lem en t m ix tu r e w as p r e s e n t# I n o r d e r t o s i m p l i f y th e r e a c t i o n m ix tu r e s i m i l a r e x p e r im e n ts w ere p erfo rm ed i n w h ic h v a r io u s c o m b in a tio n s o f t h e c o f a c t o r s w ere e m p lo y ed . The r e s u l t s r e c o r d e d i n T a b le V i l l u s t r a t e th e g e n e r a l p a t t e r n s o b ta in e d w it h t h i s ty p e o f a s s a y . R e s t in g c e l l d a ta a r e g iv e n a s a c o m p a r iso n . I t can be s e e n t h a t ev en i n t h e p r e s e n c e o f th e c o m p le te su p p le m e n t, o x y g en u p ta k e d id n o t a p p ro a c h t h a t o b ta in e d w it h r e s t i n g c e l l s . The o m m issio n o f DPN and C ytochrom e c from t h e r e a c t i o n s y s te m d id n o t d e c r e a s e o x id a t io n b elow t h e c e l l - f r e e c o n t r o l and w ou ld i n d i c a t e t h a t , i f t h e s e c o f a c t o r s a r e r e q u ir e d f o r c a p r a te o x id a t io n , t h e r e i s a s u f f i c i e n t amount o f t h e s e s u b s t a n c e s i n t h e c r u d e c e l l - f r e e e x t r a c t f o r o x id a t io n t o o c c u r . I f s u c c in a t e and r i b o - f la v in e - 5 - p h o s p h a t e w ere a l s o o m itte d ( f l a s k 3 ) th e r e w as a s l i g h t d e c r e a s e i n t h e am ount o f o x y g en consum ed a s com p a r e d t o th e c o n t r o l . I t i s p r o b a b le t h a t t h i s d e c r e a s e i n o x y g en c o n su m p tio n i s du e t o t h e a b i l i t y o f t h e e x t r a c t t o o x i d i z e s u c c i n a t e ( f l a s k 6 ) • The e s s e n t i a l f u n c t i o n o f ATP i n t h e r e a c t i o n sy s te m i s shown by t h e d a ta o b ta in e d from f l a s k s 4 and 5# R em oval o f ATP e v e n i n th e p r e s e n c e 37 TABIE IV OXIDATION OP CAPRATE AND CAPRYIATE BY CRUDE ENCE AND ABSENCE O F A CÜPACTÜR SUPPXEMENT A d d it io n t o r e a c t i o n v e s s e l S u b s t r a t e p i Og consum ed^ 30» 60* 90* 1 2 0 * 150* 180» None GlO 0 0 0 0 0 0 S u p p lem en t ^10 8 16 24 30 31 30 None % 0 0 0 0 0 0 S u p p lem en t % 3 8 9 14 22 39 1 " ' ® “ € o r r e c t i o n f o r o x y g en u p ta k e b y enzyme c o n t r o l i n th e p r e s e n c e o f a l l c o f a c t o r s bu t w ith o u t s u b s t r a t e . R e a c tio n m ix tu r e c o n ta in e d 1 ,0 ml c e l l - f r e e e x t r a c t ( 1 ,7 2 mg N ), 0 ,5 joM i n d ic a t e d s u b s t r a t e and w here in d ic a t e d 2 pM Mg++, 15 >iM GSH, 3*3 pM ATP, Q#4 jaM s u c c i n a t e , 0 ,1 mg OoA, 0 ,1 mg cy to ch ro m e c , 0 ,1 mg DPN, and 0 ,1 mg r i b o f l a v i n e - 5 ♦ -p h o s p h a te i n a t o t a l volum e o f 3 ,1 m l. I n c u b a tio n 37® C, a i r a tm o sp h e r e , 0 ,0 5 M p h o sp h a te b u f f e r , pH 7 . 1 , 0 ,1 m l 20^ KOH i n c e n t e r w e l l f o r COg a b s o r p t io n . m t S # O w rH a 0 1 o < o 6 0 OJ M # I O t . O I f I I I I I I I I r- 4 r4 r4 0 0 0 O O O o» o* c* & • o * O ï O î iH rH 60 60 H fH I I I 60 60 60 r H iH r H 0 } 0 2 03 0 2 o a $ 02 60 T f 60 6 0 0 < D + 3 (Ü U «2 0 0 9 0 Q ) 1 09 I •P 5 O * ë o o < D Q ) I I S I g ^ • < D rd 4 3 0 ( d -P ^ O p, (D ai ^ 0 O o • T I O o 38 I 09 « • » ' d o o a g i ” c d p 4 bO Q ) o g 4 » Pi ai «H 02 P i c d r H O P, rH * a i " , P H O O ^ Pi 4 3 55^0 < D O '60 4» § O 2 ^ 4 » o o X *H (D „+» P ^ ai «H o ^ rO © tO p W Pi V O o © Pi #02 O © rH © S *a 'Ci "H rH p < iH #rH Pi I 60 # O -P O ^ -P rH © O S H # G ^ g ' 1 • « • B ] > • »H o rH O O W rd 60 t> P, © P >»rH # , •H >P © Pi d *H 43 Pi © U u o o o © © # © r P ^ rH Pi © 39 o f s m a ll am ounts o f a p o s s i b l e ATP r e g e n e r a t in g s y s te m m a rk ed ly d e c r e a s e d t h e o x y g en c o n su m p tio n . The s m a ll amount o f o x y g en consum ed u n d er t h e s e c o n d it io n s may be due t o th e p r e s e n c e o f r e s i d u a l ATP i u th e cru d e e x t r a c t . P r o d u c t a n a l y s i s . The t o t a l p e r c e n t o x i d a t i o n ob t a in e d w it h t h e cru d e c e l l - f r e e sy s te m d id n o t a p p ro a c h t h a t o b ta in e d w it h r e s t i n g c e l l s . I t a p p ea red q u i t e p r o b a b le t h e r e f o r e t h a t a n in t e r m e d ia t e o f o x id a t io n was a c c u m u la tin g w h ich c o u ld n o t be f u r t h e r m e t a b o liz e d . U sin g th e cru d e c e l l - f r e e p r e p a r a tio n th e a v e r a g e o x y g en co n su m p tio n w it h c a p r y la t e a s a s u b s t r a t e was 2 ,6 Og p e r m ic r o m o le . C a lc u la t io n s b a se d on t h e c l a s s i c a l E n o o p 's b e ta o x i d a t i o n m echanism in d ic a t e d t h a t a c e t a t e o r i t s a c t i v a t e d d e r i v a t i v e was t h e a c c u m u la tin g p r o d u c t. An e x p e r im e n t was r u n t h e r e f o r e i n w h ic h t h e o x i d a t io n o f c a p r y la t e by th e cru d e e x t r a c t (su p p le m e n te d a s shown i n T a b le V I) was a llo w e d t o p r o c e e d t o c o m p le tio n . The r e a c t i o n m ix tu r e was th e n ch rom atograp h ed a s p r e v io u s l y d e s c r ib e d . Two s p o t s w ere fou n d (T a b le V I ) , one h a v in g an o f 0 ,5 4 and 0 ,5 7 , t h e o t h e r , an R^ o f a p p r o x im a te ly 0 ,4 2 , The fo rm er c o r r e sp o n d s t o a c e t a t e and e s t a b l i s h e s a c e t a t e or a c e t y l CoA a s a p r o d u c t o f th e r e a c t i o n . The l a t t e r s p o t (R^ 0 ,4 2 ) h a s n o t b een i d e n t i f i e d b u t p r o b a b ly r e p r e s e n t s some fo u r ca rb o n r e s id u e o f c a p r y la t e o x i d a t i o n . > I m t i i § M i m £h *4 g P: © I 1 4 3 © © > 4 3 g I O O © © a > © 4 3 I 0 1 © 4 3 © fH I ü © 4 3 © 4 3 © O < © © T j 3 g © * H > © 4 3 A -lO P3 O © 4 3 © U 4 3 © i I. ^ O © Î 2î g 8 O I 0 2 O c o a « o t H * 0> • O 1 0 O t 02 H * H » tO • O © 4 3 I fH H * E- lO © 4 3 i I 02 40 a © ^ P i o A W © S " , O 60 ^ ^ iH o © r H © P i ^ 4 3 *H •H © 43 d o p " , © o Pi 60 43 600 zin <© *H «H 43 3 ^ 1 © 60 O O • d 60 M ■^w . O C Q rH • c îj a rH 'd a • © ^60 • d 60 H «H rH 4-i • © O O -P .. d + © M 0 + O u A © fH ", 43 'fH 5 h X C Q © p S ",o"^ © 43 © d # 4 3 o © © © •H iH M 43 ^ d A o P i « H © © A g © © © ^ P 3 o 4 3 p « © s d s a * H 10 l O o o • d • o © o No r e s i d u a l c a p r y la t e was d e t e c t e d . IV . sm m R Y C rude c e l l - f r e e e x t r a c t s o f S . mar c e s c e n s (A lp h in ) a r e c a p a b le o f o x i d i z i n g c a p r a te and c a p r y la t e i n t h e p r e s e n c e o f a su p p lem en t m ix tu r e c o n t a in in g ATP, CoA, GSH, and Mg. I f ATP i s o m itte d o x y g en u p ta k e i s m ark ed ly d e c r e a s e d . The r e q u ir e m e n t o f th e s y s te m f o r ATP s u g g e s t s an i n i t i a l a c t i v a t i o n o f th e f a t t y a c id s w ith th e fo r m a tio n o f an a c t i v a t e d d e r i v a t i v e by a m echanism s i m i la r or i d e n t i c a l t o t h a t show n i n o t h e r s y s te m s (Lipmann e t a l . , 1 9 5 5 ) . In th e p r e s e n c e o f th e su p p lem en t th e maximum o x y g en c o n su m p tio n p e r m ic r o m o le o f s u b s t r a t e was 2*5 yM. o f o x y g e n . The o x y g en u p ta k e was n o t in c r e a s e d by th e a d d i t i o n o f cy to ch ro m e c , DPN, s u c c i n a t e , a n d /o r r i b o f l a v i n e - 5 -p h o s p h a te . Two p r o d u c ts w ere d e t e c t e d ch rom âto g r a p h ic a lly a t th e c e s s a t i o n o f c a p r y la t e o x i d a t i o n . One w as i d e n t i f i e d by i t s R f v a lu e a s b e in g a c e t a t e or i t s a c t i v a t e d d e r i v a t i v e . The se c o n d p r o d u c t d e t e c t e d by t h i s p r o c e d u r e was n o t i d e n t i f i e d , b u t a ssu m in g b e t a o x id a t io n i t i s n o t un l i k e l y t h a t t h i s p r o d u c t w ou ld b e a f o u r ca rb o n compound a t th e o x id a t io n l e v e l o f b e t a h y d ro x y b u t y r a t e . 42 Two m ajor d i f f i c u l t i e s w ere e n c o u n te r e d i n w o rk in g w it h S . m a r c e sc e n s ( A lp h in ) . I n th e f i r s t p la c e t h e o r g a n ism d id n o t grow t o o w e l l on t h e m in e r a l s a l t s c a p r a te mediumaand t h e la r g e m a sse s o f c e l l s r e q u ir e d f o r g r in d in g w ere h a rd t o o b t a in . S e c o n d ly , c e l l - f r e e e x t r a c t s p r e p a r e d from r e p l i c a t e b a tc h e s o f c e l l s , grown and p r e p a r e d u n d er e s s e n t i a l l y i d e n t i c a l c ir c u m s ta n c e s d id n o t show c o n s i s t e n t p r o p e r t i e s . In some c a s e s th e e x t r a c t s had a c t i v i t y ; i n o th e r c a s e s th e y w ere i n e r t in c a p r a te o x i d a t i o n . Con s e q u e n t ly , w ork w it h t h i s o rg a n ism w as a b a n d o n ed . CmVTER IV THE OXIDATION O P ALIPHATIC PATTY ACIDS BY CELL-FREE SYSTEMS PROM PSEUDOMONAS FLHORESCINS P .6 (1 ) PRELIMINARY STUDIES I . INTRODUCTION The e v id e n c e p r e s e n t e d i n t h e p r e v io u s c h a p te r s t r o n g l y s u g g e s t s t h a t a l i p h a t i c a c id s m ust be a c t i v a t e d p r io r t o o x id a t io n and t h a t ATP and CoA a r e in v o lv e d i n t h e a c t i v a t i o n . B e ca u se o f th e d i f f i c u l t i e s e n c o u n te r e d in^ u s in g S . m a r c e s c e n s i n c e l l - f r e e work ( s e e p r e v io u s c h a p te r ) a new o r g a n ism , P . f l u o r e s c e n s P .6 , o r i g i n a l l y i s o l a t e d from s o i l was em p loyed f o r f u r t h e r s t u d i e s . T h is o r g a n ism , i n c o n t r a s t t o th e S e r r â t i a , g a v e la r g e r c e l l y i e l d s and c o n s i s t e n t l y y i e l d e d a c t i v e c e l l - f r e e e x t r a c t s . I I . METHODS In o r d e r t o o b t a in la r g e q u a n t i t i e s o f c e l l s f o r t h e p r e p a r a tio n o f c e l l - f r e e e x t r a c t s t h e f o l l o w i n g c o n d it io n s w ere e m p lo y ed . The t e s t o r g a n ism w as grow n in 1 2 l i t e r f l a s k s c o n t a in in g 6 l i t e r s o f l i q u i d medium (pH 7 .0 ) o f t h e f o l lo w in g c o m p o s itio n : 0 .4 ^ d i s odium m a lo n a te (E a stm a n ), 0 .5 ^ KgHPO*, 0 . ^ KHaPO*, 0,1% TO^NOa, 0.1% 0,01% M g d a i n d i s t i l l e d w a t e r . I n c u b a tio n was a t room 44 te m p e r a tu r e f o r 48 h o u r s d u r in g w h ic h tim e t h e medium w as c o n t i n u a l l y a g i t a t e d w it h s t e r i l e a i r . One and o n e - h a lf p e r c e n t by volum e o f a 24 h ou r c u lt u r e p r e v io u s l y grow n i n 500 m l o f a medium o f th e sam e c o m p o s itio n s e r v e d a s t h e in o c u lu m . The c e l l s w ere h a r v e s t e d w it h a S h a r p ie s c e n t r i fu g e ( 1 2 ,0 0 0 RPM), th e com pact c e l l m ass b e in g r e su sp e n d e d i n c o ld d i s t i l l e d w a te r and c e n t r if u g e d i n th e c o ld (4®C t 1^ 0) a t 2 0 ,0 0 0 x g . T h is p r o c e s s w as r e p e a t e d u n t i l t h e c e l l s had b e e n w ashed w it h 10 v olu m es p e r c e n t o f d i s t i l l e d w a te r i n r e s p e c t t o t h e o r i g i n a l m edium . The r e s u l t i n g c e l l p a s t e was th e n t h i n l y sp r e a d i n a p e t r i d i s h . The p e t r i d is h was p la c e d i n a ,d e s i c c a t o r and d r ie d in v a cu o ( 2 - 5 m m Hg) o v er D r i e r i t e a t room te m p e r a tu r e . D ry c e l l s n o t u s e d im m e d ia te ly f o r t h e p r e p a r a tio n o f c e l l - f r e e e x t r a c t s w ere s t o r e d o v e r d e s ic c a n t i n th e r e f r i g e r a t o r . P r e p a r a t io n o f c e l l - f r e e e x t r a c t . One t o 1 .5 p a r t s o f c o ld 0 .0 5 M p h o sp h a te b u f f e r (pH 6 .1 ) w ere ad d ed t o a g iv e n q u a n t it y o f d r ie d c e l l s c o n ta in e d w i t h in a m o rta r w h ic h was p r e v io u s ly f r o z e n i n i c e . The i n g r e d ie n t s w e re th e n l i g h t l y ground as 5 p a r t s o f ground g l a s s ( f i n e g r a d e )I I w ere s lo w ly a d d ed . . The m ix tu r e w as ground w it h maximum | hand p r e s s u r e f o r t h r e e 1 m in u te i n t e r v a l s . B etw een e a ch g r in d in g th e m ix tu r e w as sc r a p e d f r e e o f th e m ortar t o w h ic h i t had f r o z e n . A t th e end o f th e g r in d in g p e r io d th e ~45 cream y p a s t e w as r e su sp e n d e d and e x t r a c t e d w ith . 20 p a r t s o f c o ld 0 .0 5 M p h o sp h a te b u f f e r (pH 6 . 1 ) , and c e n t r if u g e d a t 2 0 ,0 0 0 X g f o r 50 m in u te s i n th e c o ld (4®C t 1®C) • The r e s u l t i n g c le a r s u p e r n a ta n t w as th e n e i t h e r u s e d im m e d ia te ly or s t o r e d i n s c r e w -to p t e s t tu b e s a t - 1 5 ° G u n t i l n e e d e d . Su ch c e l l - f r e e p r e p a r a tio n s h a v e b e e n s t o r e d f o r a p e r io d o f s e v e r a l w eek s w it h l i t t l e l o s s o f a c t i v i t y . M ethods f o r p r o d u c t a n a l y s i s and m an om etric d e t e r m in a tio n s f o r o x y g en u p ta k e h a v e p r e v io u s ly b e e n d e s c r i b e d , I I I . EXPERIMENTAL RESULTS C o fa c to r r e q u ir e m e n ts . Crude c e l l - f r e e e x t r a c t s o f P , f l u o r e s c e n s P . 6 w ere t e s t e d f o r t h e i r a b i l i t y t o o x i d i z e c a p r a te i n t h e p r e s e n c e and a b s e n c e o f th e su p p le m e n ts a l r e a d y im p lic a t e d a s b e in g r e q u ir e d from t h e work w it h § . m a r c e s c e n s ( A lp h in ) , As w it h t h e S e r r â t i a e x t r a c t s , th e p ,6 e x t r a c t s d id n o t o x i d i z e c a p r a te or c a p r y la t e u n l e s s su p p lem en ted (T a b le V I I ) . In t h e p r e s e n c e o f Mg, GSH, GoA, and ATP a b o u t 1 ^ o f o x y g en w as consum ed p e r m icro m o le o f s u b s t r a t e , a v a lu e c o n s id e r a b ly l e s s th a n t h e 2 .5 t o 5 .0 fo u n d w it h t h e S e r r â t l a e x t r a c t s . A c e t a t e w h ich w ould be form ed a t th e 1 jiM l e v e l a c c o r d in g t o t h e b e t a o x i d a t i o n m echanism w as n o t o x id iz e d e v e n i n t h e p r e s e n c e o f th e su p p le m e n t. 46] TABLE VII OXIDATION OP CAPRATE AND ACETATE BY CRUDE CELL-PH^ EXTRACTS OP P . PLUORESCENS P . 6 IN THE PRESENCE AND ABS5NCS Ô F T 'Û'Î^CTOR SUPPIEMENT A d d it io n t o S u b s t r a t e )a 1 Og consumed*” * r e a c t i o n " ......... v e s s e l 30» 60» 90» 1 2 0 » 150» 180» 210» None 2 pM G io 0 0 0 0 0 0 0 su p p le m e n t 2 ) M CiQ 8 12 18 23 30 4 0 46 None 8 pM Gg 0 0 0 0 0 0 0 S u p p lem en t 8 pM Gg 0 0 0 0 0 0 0 None 2 pM G q 0 0 0 0 0 0 0 su p p lem en t 2 pM Ca 11 23 23 25 23 - - ■ ^ ‘ ‘ C o r r e c te d f o r o x y g e n u p ta k e b y enzym e c o n t r o l i n a b s e n c e o f s u b s t r a t e . R e a c t io n m ix tu r e c o n ta in e d 0 .1 m l cru d e c e l l - f r e e e x t r a c t , ( 0 .4 1 mg N ), c a p r a t e , c a p r y la t e or a c e t a t e a s s u b s t r a t e , and w here i n d ic a t e d , a su p p lem en t c o n t a in in g 2 >iM M g '^ " * ’, 13 GSH, 3 . 3 pM ATP, 33 u n i t s CoA i n a t o t a l volu m e o f 2 .1 m l. I n c u b a tio n 30 G, a i r a tm o sp h e r e , 0 ,0 5 p h o sp h a te b u f f e r , pH 6 , 1 . 0 .1 m l 20^ KOH i n c e n t e r w e l l f o r COg a b s o r p t io n . E s s e n t i a l n a tu r e o f Co enzyme A and ATP. When ATP was o m itte d from t h e r e a c t i o n sy s te m no o x y g e n co n su m p tio n was o b se r v e d (T a b le V I I I ) i n d i c a t i n g t h e r e q u ir e m e n t o f th e s y s te m f o r t h i s com pound. When C O A a lo n e w as o m itte d t h e r e w as a s m a ll amount o f o x y g e n consum ed b u t o n e w h ich w as l e s s th a n i n t h e ATP-CoA su p p lem en ted r e a c t i o n v e s s e l s (T a b le V I I I ) . T h is l a t t e r o b s e r v a t io n s u g g e s t e d t h a t t h e r e was a CoA c o n ta m in a tio n i n th e c r u d e c e l l - f r e e s y s te m . T h e r e fo r e , t h e cru d e e x t r a c t w as t r e a t e d w i t h D ow ex- 1 -h y d r o c h i or id e a c c o r d in g t o t h e m ethod o f sta d tm a n e t a l . , (1 9 5 1 ) w h ic h e f f e c t i v e l y r e d u c e s th e CoA l e v e l o f enzym e p r e p a r a t io n s . The Dowex t r e a t e d e x t r a c t was r e -e x a m in e d f o r CoA d e p e n d e n c y . The r e a c t i o n m ix tu r e c o n ta in e d ATP, GSH, and Mg, w it h and w ith o u t CoA. CoA was u s e d a t a l e v e l o f 55 u n i t s w h ic h i s e q u iv a le n t t o 0 .1 pM c a lc u l a t e d a c c o r d in g t o th e m o le c u la r w e ig h t r e p o r te d by N o v e l l i ( 1 9 6 3 ) . T a b le IX show s t h a t t h e r e was no o x y g en co n su m p tio n by th e t r e a t e d e x t r a c t i n th e a b s e n c e o f ad d ed CoA, w h e r e a s o x i d a t io n w as r e s t o r e d on th e a d d i t io n o f 0 .1 pM CoA. T h e se d a ta d e f i n i t e l y show t h a t CoA and ATP a r e i n v o lv e d i n a p rim a ry r e a c t i o n w h ic h p r e c e d e s o x id a t io n th u s e s t a b l i s h i n g t h e i n i t i a l s t e p i n th e c a ta b o lis m o f c a p r a te by t h i s o rg a n ism a s b e in g an ATP-CoA d ep en d en t a c t i v a t i o n . T h is s i t u a t i o n i s a n a lo g o u s t o t h e m e ta b o lism o f o th e r 48 TASm V I I I EFFECT OF ATP AND C O A O N CAPRATE OXIDATION BY A CRUDE CELL-FREE EXTRACT OF P . PLUORESCENS P . 6 A d d it io n s t o r e a c t i o n m ix tu r e 1 Og consum ed* 30» 60» 90» 1 2 0 » 150» 180» ATP, COA, GSH, Mg 8 12 18 23 30 4 0 ATP, GSH, M g 1 .5 6 10 6 12 18 COA, GSH, Mg 0 0 2 4 3 0 GSH, Mg 0 0 0 0 0 0 ‘ " ‘C o r r e c te d f o r oxy g en u p ta k e by enzyme c o n t r o l i n a b s e n c e o f s u b s t r a t e . R e a c t io n m ix tu r e c o n ta in e d 0 .1 m l cru d e c e l l - f r e e e x t r a c t ( 0 .6 2 mg N ), 2 pM c a p r a te and w h ere i n d ic a t e d 2 pM Mg++ , 0 .1 pM GoA, 3 .3 pM ATP, 1 3 pM GSH i n a t o t a l volum e o f 2 .1 m l. I n c u b a t io n 3 0° C, a i r a tm o sp h e r e , 0 .0 5 M p h o s p h a te b u f f e r , pH 6 . 1 . 0 .1 m l 20^ KOH i n c e n t e r w e l l f o r COg a b s o r p t io n . 49 TABI2 D C EFFECT OF CO A ON CAPRATE OXIDATION BY A DOWEX-1- HYDROCHLORIDB TREATED CELL-FREE EXTRACT OF P . PLUORESCENS P . 6 A d d it io n s t o p i Og consum ed’ * r e a c t i o n m ix tu r e ----------------------------------------------- 30» 60» 90» 1 2 0 » 150» 180» 2 1 0 » ATP, COA, GSH, M g*'"' 8 12 18 23 30 4 0 46 ATP, GSH, M g " * " * ' 0 0 0 0 0 0 0 ATP, CoA, GSH, Mg 12 22 31 39 4 2 45 49 ^ C o r r e c te d f o r o x y g en u p ta k e by enzyme c o n t r o l in a b s e n c e o f s u b s t r a t e . * * C o n tr o l f l a s k c o n t a in in g cru d e (n o t Dowex t r e a t e d ) c e l l - f r e e e x t r a c t . R e a c t io n m ix tu r e c o n ta in e d 0 .1 m l Dowex t r e a t e d c e l l f r e e e x t r a c t ( 0 .0 6 m g .N ), 2,pM c a p r a te and w here in d ic a t e d 2 pM M g'*"*', 0 .1 pM G O A , 3 .3 pM ATP, 13 pM GSH i n a t o t a l volu m e o f 2 .1 m l. I n c u b a tio n 3 0 ° C, a i r a tm o sp h e r e , 0 .0 5 M p h o sp h a te b u f f e r , pH 6 . 1 . . 0 .1 m l 20^ KO H i n c e n te r w e l l f o r COg a b s o r p t io n . s o ’ f a t t y a c id s o c c u r r in g w i t h m am m alian t i s s u e and b a c t e r i a l s y s t e m s . D e t e c t io n o f a c e t a t e . The i n a b i l i t y o f th e cru d e c e l l - f r e e e x t r a c t t o o x i d i z e a c e t a t e (T a b le V I I) c o u p le d w it h th e f a ç t t h a t t h e same e x t r a c t consum ed o n ly 1 pM o f o x y g en w it h c a p r a te a s t h e s u b s t r a t e s u g g e s t e d t h a t a c e t a t e or a c e t y l GoA w ou ld be a p r o d u c t o f th e r e a c t i o n . To t e s t t h i s p o s s i b i l i t y r e a c t i o n v e s s e l s w e re s e t up i n w h ic h cru d e e x t r a c t was a llo w e d t o o x i d i z e c a p r a te i n th e p r e s e n c e o f th e c o m p le t e .su p p le m e n t. A t t h e c e s s a t i o n o f o x y g en u p ta k e , th e r e a c t i o n m ix tu r e was e s t e r i f i e d w it h e t h y l a l c o h o l . and t r e a t e d w it h h y d r o x y la m in e a s p r e v io u s l y d e s c r ib e d . The r e s u l t i n g h yd roxam ic a c id s w ere t h e n ch ro m a to g ra p h ed a lo n g w i t h c o n t r o l s f o r a c e t a t e , c a p r a t e , and c a p r y la t e . The r e s u l t s a r e r e c o r d e d i n T a b le X . Two s p o t s w ere d e t e c t e d by t h i s p r o c e d u r e ; a c e to h y d r o x a m a te w h ic h was i d e n t i f i e d b y i t s v a lu e and a se c o n d s p o t a p p e a r in g i n one o f t h e e x p e r im e n ts h a v in g a n R^ o f 0 .3 5 w h ic h was n o t i d e n t i f i e d , s i n c e e s t é r i f i c a t i o n was c a r r ie d o u t p r io r t o th e h yd roxam ate f o r m a t io n , i t c a n n o t be s t a t e d a t t h i s tim e a s t o ,w h e t h e r a c e t a t e or a c t i v e a c e t a t e was p r e s e n t a t t h e end o f t h e r e a c t i o n . S u r p r i s in g ly , no s p o t c o r r e s p o n d in g t o c a p r y la t e w as o b s e r v e d . T h is may h a v e b e e n due t o t h e am ount o f t h i s compound i n t h e r e a c t i o n 5l^ TABm X Rf. VALUES O P OAPRATE OXIDATION PRODUCTS OP CRUDE CELL-FREE EXTRACTS C F P . FLU ORES CENS P . 6 F la s k R f v a lu e s R^ v a lu e s o f c o m p a r a tiv e sta n d a r d s N o. o b ta in e d ' — ' - ........................ — ------------------------- A c e t a t e C a p r y la te C a p ra te 1 0 .6 3 0 .6 2 - 0 .6 5 0 .9 4 - 0 .9 7 0 .9 6 - 0 .9 8 2 0 .6 2 ^ 0 .3 5 R e a c t io n m ix tu r e c o n ta in e d 0 .1 m l cru d e c e l l - f r e e e x t r a c t ( 0 .6 2 mg N ), 2 pM c a p r a te , 2 pM Mg+^, 0 .1 pM CoA, 3 .3 uM ATP, 13 uM GSH i n a t o t a l volum e o f 2 .1 m l. I n c u b a tio n 30 ° G, a i r a tm o sp h e r e , 0 .0 5 M p h o sp h a te b u f f e r , pH 6 . 1 . 0 .1 m l 20^ KOH i n c e n t e r w e l l f o r COg a b s o r p t io n . C o n tr o l f l a s k s w ith o u t s u b s t r a t e show ed no s p o t s . I 52 ! v e s s e l s i n c e c o n t r o l s p o t s u s i n g s m a ll am ounts o f c a p r y la t e a r e v e r y f a i n t . IV . SUM M ARY AND DISCUSSION Crude c e l l - f r e e e x t r a c t s o f P . f l u o r e s c e n s P . 6 co n sumed 1 o f o x y g en p e r m icro m o le o f c a p r a te when su p p le m e n te d w it h ATP, COA, GSH, and Mg. T h is l e v e l o f o x i d a t io n c o u ld n o t b e in c r e a s e d by t h e a d d i t io n o f v a r io u s c o f a c t o r s . I n th e a b s e n c e o f any added ATP t h e o x y g e n conS su m p tio n was n i l d e m o n s tr a tin g t h e n e c e s s i t y o f t h i s c o - f a c t o r f o r o x i d a t i o n . D o w ex -l-H C l t r e a t e d cru d e c e l l - f r e e e x t r a c t su p p lem en ted w it h ATP, GSH and Mg d id n o t o x id iz e c a p r a te u n l e s s CoA w as a l s o a d d e d . The d a ta show t h a t an ATP and CoA a c t i v a t i o n o f th e f r e e a c id w as n e c e s s a r y fo r m in g c a p r y l GoA w h ic h a c t e d a s t h e s u b s t r a t e f o r o x i d a t io n r a t h e r th a n c a p r a t e . A c e t a t e was n o t o x id iz e d b y t h e cru d e e x t r a c t w it h AT?, COA, GSH and Mg a s su p p le m e n ts . C h rom atograp h ic e v i d e n c e o f a c e t a t e (o r a c t i v e a c e t a t e ) a s a p r o d u c t when o n ly 1 o f oxy g en was consum ed p e r m icr o m o le o f c a p r a te e li m in a t e s m u l t i p l e a l t e r n a t e and omega o x i d a t i o n a s p o s s i b l e m ech a n ism s, and o n ly b e t a o x i d a t i o n ca n a c c o u n t f o r a c e t a t e a c c u m u la tio n a t t h i s o x id a t io n l e v e l . CHAPTER V THE OXIDATION OF ALIPHATIC FATTY ACIDS BY CELL-FREE SYSTEMS FROM PSEDDOMONAS FLUORmCENS P . 6 (2 ) FRACTIOHATIOH OF CELL-FREE EXTRACTS AND PROPERTIES OF THE PARTIALLY PURIFIED ENZYME SYSTEM I . INTRODUCTION F or f u r t h e r i n v e s t i g a t i o n s i t was d e s i r a b l e t o a t tem p t f u r t h e r p u r i f i c a t i o n o f th e enzyme p r e p a r a t io n , su c h p u r i f i c a t i o n w ou ld f a c i l i t a t e th e tr a p p in g o f th e a c t i v a t e d d e r i v a t i v e o f c a p r a te and a llo w a more d e t a i l e d i n v e s t i g a t i o n o f t h e o x id a t io n r e a c t i o n s . I I . METHODS I d e n t i f i c a t i o n o f th e end p r o d u c ts o f th e r e a c t i o n w as a c c o m p lis h e d by ch ro m a to g ra p h in g t h e compounds a s th e f r e e a c id s a f t e r th e m ethod o f Lugg and O v e r e ll ( 1 9 4 8 ) , K eto a c id s w ere d e te r m in e d by r e a c t i o n w it h 2 , 4 - d i n i t r o - p h e n y lh y d r a z i n e . I I I . EXPERIMENTAL RESULTS ! Ammonium s u l f a t e f r a c t i o n a t i o n . C e l l - f r e e e x t r a c t s w ere o b ta in e d a s p r e v io u s l y d e s c r ib e d . Pow dered ammonium s u l f a t e w as added t o a g iv e n volum e o f t h e c e l l - f r e e 54 j e x t r a c t t o o b t a in 20 p e r c e n t s a t u r a t i o n , th e q u a n t i t i e s : f i g u r e d a c c o r d in g t o th e nomogram o f D ix o n ( 1 9 5 5 ) . The r e s u l t i n g p r e c i p i t a t e was c o l l e c t e d b y c e n t r i f u g a t i o n f o r 30 m in u te s a t 2 0 ,0 0 0 x g . The s u p e r n a ta n t was d e c a n te d and th e p r e c i p i t a t e d r a in e d by i n v e r t i n g i t s c o n t a in e r on a b s o r b e n t p a p er t o w e lin g . The p r e c i p i t a t e w as r e d i s s o l v e d in 10 m l o f d i s t i l l e d w a te r * The s u p e r n a ta n t was th e n b r o u g h t t o 4 0 p e r c e n t s a t u r a t i o n and th e p r o c e d u r e r e p e a te d u n t i l ammonium s u l f a t e p r e c i p i t a t e d f r a c t i o n s o f 0 - 2 0 , 2 0 -4 0 , 4 0 - 6 0 , 6 0 - 8 0 , and 8 0 -1 0 0 p er c e n t s a t u r a t i o n w ere o b t a in e d . A l l m a n ip u la tio n s w e r e c a r r ie d o u t in t h e c o ld (4°G t 1°C ) w it h th e e x c e p t io n o f th e s a l t i n g o u t p r o c e d u r e w h ic h was c a r r ie d o u t a t room te m p e r a tu r e . The pH o f t h e r e d i s s o l v e d p r e c i p i t a t e s w as b e tw e e n 5 . 5 and 6 . 0 . The 3 2 0 -4 0 f r a c t i o n o b ta in e d b y t h i s p r o c e d u r e was o p a l e s c e n t w it h a f a i n t g r e e n t i n g e w h erea s t h e S4 0 - 6 O f r a c t i o n was c h a r a c t e r iz e d by a d i s t i n c t r e d d ish -b r o w n h u e . The o x id a s e a c t i v i t y o f t h e v a r io u s f r a c t i o n s w as c h e ck ed im m e d ia te ly a f t e r p r e p a r a tio n b y m an om etric m eth od s p r e v io u s ly d e s c r ib e d . The o x i d i z i n g a b i l i t y o f t h e p u r ifie d , c e l l - f r e e e x t r a c t a g a i n s t c a p r a te was l o c a t e d i n t h e S40 - 6 O f r a c t i o n (T a b le X I) w h er ea s th e S 20-.40 f r a c t i o n c o n ta in e d a c t i v i t y to w a r d s m a lo n a te (W o lfe , 1 9 5 4 ) . I t can a l s o be s e e n (T a b le X I) t h a t t h e o x y g en consum ed p e r m ole o f TABIE X I CAPRATE OXIDATION BY REDISSOLVED AM M ONIUM SULFATE PRECIPITATED FRACTIONS 0F A CELL-FREE EXTRACT OF P , FLUORES CENS, P .G % Ammonium s u l f a t e s a t u r a t i o n p i Og consumed*'®’ 210* 240* 270» 300» 330» 360» 0 -2 0 non e 2 0 -4 0 n on e 4 0 -6 0 0 6 , 3 8 , 8 1 6 .6 20 20 6 0 -8 0 non e 8 0 -1 0 0 non e ^ C o r r e c te d f o r oxygen u p ta k e by enzyme c o n t r o l in a b s e n c e o f s u b s t r a t e . R e a c t io n m ix tu r e c o n t a in e d 0 .1 m l o f t h e r e d i s s o l v e d p r e c i p i t a t e , 2 ^ c a p r a t e , 35 u n i t s CoA, 13 pM GSH, 3 ,3 pM ATP and 2 M g '* " * " i h a t o t a l volum e o f 2 ,1 m l. I n c u b a tio n 3 0 ° G, a i r a tm o sp h e r e , 0 ,0 5 M p h o sp h a te b u f f e r , pH 6 , 1 , 0 ,1 m l 20^ KOH i n c e n t e r w e l l f o r COg a b s o r p t io n . 56l c a p r a te was a p p r o x im a te ly h a l f t h a t consum ed w it h th e cru d e c e l l - f r e e e x t r a c t . C h rom atograph ic and c h e m ic a l t e s t s show ed t h e a b s e n c e o f a c e t a t e and k e t o a c id s b u t r e v e a le d an a s y e t u n i d e n t i f i e d compound w it h an o f 0 .2 2 . T h is c o u ld m ean t h a t e i t h e r som e c o f a c t o r was i n l i m i t i n g am ount or t h a t o x i d a t i o n was b e in g b lo c k e d ab ove th e c le a v a g e o f a c e t a t e or a c e t y l GoA. I n a d d i t io n i t sh o u ld be n o te d t h a t a lo n g l a g o c c u r r e d p r e v io u s to o x y g e n u p ta k e . An e x p la n a t io n f o r t h i s phenom enon i s d i s c u s s e d l a t e r . O x id iz in g c a p a c it y o f t h e S4 0 - 6 O f r a c t i o n . I n o r d e r t o g a in a f u r t h e r i n s i g h t in t o t h e o x i d a t i v e p r o p e r t i e s o f th e S 4 0 - 6 O f r a c t i o n , th e a b i l i t y o f t h i s f r a c t i o n t o o x i d i z e c a p r a te and o th e r s u b s t r a t e s i n t h e p r e s e n c e o f v a r io u s c o f a c t o r s was t e s t e d . T a b le X II su m m arizes th e d a ta o b ta in e d i n t h e s e e x p e r im e n ts . T h ese d a ta i n d i c a t e t h a t th e a d d i t io n o f TPN t o t h e sta n d a r d su p p le m e n t had no e f f e c t on th e t o t a l o x y g e n consum ed when c a p r a te was th e s u b s t r a t e . B e ta k e t o c a p r a t# was n o t o x id iz e d u n d er t h e e x p e r im e n ta l c o n d i t i o n s . A c e t a t e w as n o t o x id iz e d b y t h i s sy s te m u n le s s a su p p lem en t c o n t a in in g o x a l a c e t a t e and TPN a s w e l l a s t h e o th e r sta n d a r d c o f a c t o r s w as add ed t o th e r e a c t i o n m ix t u r e . T h is o b s e r v a t io n in d ic a t e d t h e p r e s e n c e o f t h e c i t r a t e c o n d e n s in g enzym e and a t l e a s t one su b s e q u e n t o x i d a t i v e r e a c t i o n . The co n su m p tio n o f o n ly 1 ^ 57 TABLE X I I OXIDIZING CAPACITY OP THE S4 0 - 6 O FRACTION A d d it io n s t o r e a c t i o n m ix tu r e p i Og consum ed i n 1 8 0 » * A c e t a t e 16 pM c i s - A c o n i t a t e 16 pM C a p ra te 10 pM B e t a - k e t o c a p r a te 2 pM ATP, COA, GSH, M g 0 0 11 0 ATP, COA, GSH, Mg, TPN 0 0 11 0 ATP, GOA, GSH, Mg, O A C 2 0 - - ATP, COA, GSH, Mg, TPN, O A C 17 0 - _ ''^Corrected f o r o x y g en u p ta k e by enzym e c o n t r o l i n a b s e n c e o f a c e t a t e , c i s - a c o n i t a t e , c a p r a te , and b e t a - k e t o c a p r a t e . R e a c t io n m ix tu r e c o n t a in e d i n d ic a t e d s u b s t r a t e , 0 ,1 m l S4 0 - 6 O f r a c t i o n ( 0 ,0 7 mg N ), 0 ,1 pM CoA, 13 pM GSH, 3 ,3 pM ATP, 2 pM Mg++, and w h ere add ed 0 ,2 mg TPN, and 20 pM o x a l a c e t a t e in a t o t a l volum e o f 2 ,1 m l. I n c u b a tio n 3 0° C, a i r a tm o sp h e r e , 0 ,0 5 M p h o sp h a te b u f f e r , pH 6 , 1 , 20^ KOH i n c e n t e r w e l l f o r COg a b s o r p t io n . 0 ,1 m l I " 58 j o f o x y g en r a th e r th a n th e e x p e c te d 8 pM I n th e o x id a t io n o f I c i t r a t e form ed from t h e 16 pM a c e t a t e o x a l a c e t a t e c o n d e n sa t i o n i s p r o b a b ly due t o t h e lo w l e v e l o f TPN p r e s e n t i n th e r e a c t i o n m ix tu r e . The e f f e c t o f f l u o r i d e on th e o x id a t io n o f c a p r a te by th e c e l l - f r e e sy ste m w as n e x t i n v e s t i g a t e d . I t w as n o te d t h a t th e a d d i t io n o f 100 pM o f f l u o r i d e t o th e r e a c t i o n m ix tu r e r e s u l t e d i n a d e c r e a s e in t h e le n g t h o f t h e la g p e r io d and a l s o in a s l i g h t in c r e a s e i n th e o x y g en c o n sumed (T a b le X I I I ) . S in c e ATP was i n l i m i t i n g c o n c e n tr a t i o n i n t h i s e x p e r im e n t, t h e in c r e a s e d o x y g en u p ta k e , i f s i g n i f i c a n t , may h ave b e e n due to th e i n h i b i t i o n by f l u o r i d e o f some r e a c t i o n c o m p e tin g f o r th e ATP. The d e y c r e a s e i n t h e l a g p e r io d s u g g e s t s t h a t some i n h i b i t o r y f a c t o r w as b e in g a l e v i a t e d by th e a d d i t io n o f f l u o r i d e . F lu o r id e i s known t o i n h i b i t e n o la s e and o th e r m agnesium d ep en d en t s y s te m s (W arburg and C h r is t ia n , 1 9 4 1 ) by c o m p le x in g w it h t h e Mg i n t h e p r e s e n c e o f p h o sp h a te t o fo rm a flu o r id e -m a g n e s iu m -p h o s p h a te c o m p le x . T h is s u g g e s t e d t h a t Mg c o u ld be p r e s e n t i n i n h i b i t o r y c o n c e n t r a t io n i n t h e r e a c t i o n m ix tu r e and i t s e f f e c t i v e c o n c e n t r a t io n was b e in g r e d u c e d by t h e ad d ed f l u o r i d e . T h is p o s s i b i l i t y was t h e r e f o r e i n v e s t i g a t e d . 59 TABLE X I I I THE EFFECT OF FLUORIDE ON CAPRATE OXIDATION BY THE S40 - 6O FRACTION A d d it io n s t o r e a c t i o n m ix tu r e >^1 Og consum ed’ * ^ 180» 210* 240» 270* 300* 330» None 0 7 9 10 18 - 100 pM Nap 6 12 16 - 20 29 '^ C orrected f o r o x y g en u p ta k e by enzyme c o n t r o l in a b s e n c e o f s u b s t r a t e . R e a c t io n m ix tu r e c o n ta in e d 0 ,1 m l f r a c t i o n (D75 mg N ), 5 pM c a p r a t e , 0 ,1 pM COA, 15 pm GSS, 3 ,3 pM ATP, 2 pM M g*^ *^ , and NaF a s in d ic a t e d i n a t o t a l volu m e o f 2 ,1 m l. I n c u b a tio n 30° C, a i r a tm o sp h e r e , 0 ,0 5 M p h o sp h a te b u f f e r , pH 6 , 1 , 0 ,1 m l 20g& KOH i n c e n t e r w e l l f o r COg a b s o r p t io n . M agnesitua e f f e c t on a d ia ly z e d S40- 6O p r e p a r a t io n . F iv e m i l l i l i t e r s o f t h e p a r t i a l l y p u r i f i e d enzym e w ere d ia ly z e d i n th e c o ld (4°G Ï 1°C ) a g a i n s t 2 l i t e r s o f d i s t i l l e d w a t e r . A f t e r 12 h o u rs d i a l y s i s , th e d i a l y z i n g medium was ch an ged and t h e d i a l y s i s c a r r ie d o u t f o r an ad d i t i o n a l 12 h o u r s . The d i a l y z e d p r e p a r a tio n was t h e n c h e ck ed f o r i t s a b i l i t y t o o x i d i z e c a p r a te i n th e p r e s e n c e o f ATP, CoA and GSH, w it h and w ith o u t m agn esiu m . The d a ta o b ta in e d a r e p r e s e n t e d i n F ig u r e 6 , I t c a n b e s e e n t h a t i n th e a b s e n c e o f added mag n e siu m , o x id a t io n o f c a p r a te w as im m ed ia te w h erea s an ap p r e c i a b l e la g o c c u r r e d upon th e a d d it io n o f t h i s c a t i o n t o t h e r e a c t i o n m ix t u r e . The maximum r a t e o f o x id a t io n was g r e a t e r i n t h e a b s e n c e o f ad d ed Mg, b u t oxy g en u p ta k e p e r m o le o f s u b s t r a t e w as e s s e n t i a l l y th e sam e. T h ese d a ta show t h a t th e f a t t y a c id o x i d i z i n g system^ i s v e r y s e n s i t i v e t o th e Mg c o n c e n t r a t io n and i t i s ap p a r e n t t h a t th e a d d i t io n o f 2 pM o f m agnesium i n some manner te m p o r a r ily i n h i b i t s t h e o x i d a t i o n . The e x a c t n a tu r e o f t h i s i n h i b i t i o n and i t s a p p a r e n t r e l i e f a f t e r a p p r o x im a te ly 2 h o u r s h a s n o t a s y è t b een f u r t h e r i n v e s t i g a t e d . W hether or n o t Mg i s r e q u ir e d f o r e i t h e r a c t i v a t i o n o r o x id a t io n c a n n o t be d e c id e d u n e q u iv o c a b ly from t h e s e 5 50 120 180 240 MINUTES 500 360 L egend: # “ 2 pM c a p r a t e , no m agnesium ; ® = 4 pM c a p r a t e , no m agnesium ; ^ = 2 p M c a p r a t e , 2 pM m agnesium . R e a c t io n m ix tu r e c o n ta in e d 0 ,1 m l d ia ly z e d S40 - 6 O f r a c t i o n ( ,0 7 3 mg N ), 0 ,1 pM CoA, 13 pM GSH, 3 ,3 pM ATP, 5 pM KgHPO* and w here i n d ic a t e d , 2 pM Mg i n a t o t a l volu m e p f 2 ,1 m l. I n c u b a tio n 3 0 ° C, a i r a tm o sp h e r e , 0 ,0 1 M T r is b u f f e r , pH 7 , 4 . 0 , 1 m l, 20^ KOH i n c e n t e r w e l l f o r COg a b s o r p t io n . C o r r e c te d f o r o x y g en u p ta k e by enzym e c o n t r o l i n a b s e n c e o f s u b s t r a t e . FIGURE 6 EFFECT OF MAGNESIUM ON CAPRATE OXIDATION BY A DIALYZED S4 0 - 6O FRACTION 62 r e s u l t s . A lth o u g h t h e d a ta s t r o n g l y s u g g e s t t h a t t h e r e i s no M g r e q u ir e m e n t f o r o x i d a t i o n , p e r s e . I f m agnesium i s r e q u ir e d in t h e a c t i v a t i o n r e a c t i o n t h i s r e q u ir e m e n t was m et by t r a c e s o f Mg l e f t i n th e d ia ly z e d p r e p a r a t io n . I t c o u ld b e t h a t a s t a b l e M g-enzyme com p lex e x i s t s w h ic h i s n o t b r o k e n by d i a l y s i s a lth o u g h no d a ta a r e a v a i l a b l e on t h i s p o i n t , IV . SUM M ARY A 4 0 -6 0 p e r c e n t ammonium s u l f a t e f r a c t i o n a t i o n o f th e cru d e c e l l - f r e e e x t r a c t o f P , f l u o r e s c e n s P .6 , c o n t a in e d th e f a t t y a c id o x id a s e s y s te m s f o r c a p r a t e . The p u r if i e d p r e p a r a tio n consum ed o n ly 0 ,6 pM o x y g en p e r m ic r o m ole c a p r a t e , G h rom atograp h ic and c h e m ic a l t e s t s showed the: a b s e n c e o f a c e t a t e and k e t o a c id s an d r e v e a le d an a s y e t u n i d e n t i f i e d compound w it h an R f o f 0 ,2 2 , A c e t a t e was o x id iz e d by t h e S40 - 6 O f r a c t i o n o n ly when o x a l a c e t a t e and TPN w ere add ed t o th e r e a c t i o n m ix tu r e d e m o n s tr a tin g th e p r e s e n c e o f t h e c o n d e n s in g enzym e and a t l e a s t one o x i d a t i v e s t e p . F lu o r i d e , an e s t a b l i s h e d i n h i b i t o r o f M g d ep e n d en t r e a c t i o n s , s t im u la t e d th e o x id a t io n o f c a p r a t e . A n a ly s is o f th e Mg e f f e c t o f a d ia ly z e d S40 - 6O f r a c t i o n show ed t h a t t h e a d d i t io n o f 2 pM o f m a g n esiu m .t o th e r e a c t i o n sy ste m 63 i n h i b i t e d t h e r a t e o f o x id a t io n and c a u s e d a tem p o ra ry i n h i b i t i o n o f th e o x i d a t i o n . T h is i n h i b i t i o n was r e l i e v e d a f t e r a p p r o x im a te ly 120 m in u te s . From t h e s e r e s u l t s , i t w as i n d ic a t e d t h a t i f a m agnesium r e q u ir e m e n t e x i s t s f o r th e a c t i v a t i o n a n d /o r o x i d a t i o n i t i s m et by t r a c e s o f t h i s c a t i o n i n th e d i a l y z e d p r e p a r a t io n . CHAPTER VI THE OXIDATION O P ALIPHATIC PATTY ACIDS BY CELL-FREE SYSTEMS PROM PSEUDOMONAS PLUORESCENS P .6 (3 ) QUALITATIVE AND QUANTITATIVE TRAPPING OP ACTIVATED CAPRATE I . INTRODUCTION The e s t a b lis h m e n t o f an ATP and ^CoA d ep en d en ce f o r t h e o x i d a t i o n o f c a p r a te (C h a p ter IV ) s u g g e s t e d t h a t th e i n i t i a l s t e p in f a j j t y a c id o x i d a t i o n was an a c t i v a t i o n o f th e c a p r a te m o le c u le . Q u a l i t a t i v e and q u a n t i t a t i v e t r a p p in g e x p e r im e n ts w ere t h e r e f o r e r u n i n an a tte m p t t o e s t a b l i s h c a p r y l CoA a s th e a c t i v a t e d p r o d u c t . I I , METHCDS The p r e p a r a tio n o f th e 8 4 0 -6 0 f r a c t i o n h as p r e v i o u s l y b een d e s c r ib e d . The d e t e c t i o n o f b i o l o g i c a l l y form ed e s t e r s w as c a r r ie d o u t i n tr a p p in g e x p e r im e n ts in w h ic h h y d r o x y la m in e was in c u b a te d d i r e c t l y w ith th e r e a c t i o n m ix tu r e (Lipm ann and T u t t l e , 1 9 5 0 ) , F o r q u a l i t a t i v e d e t e r m in a t io n s th e h yd roxam ic a c id s w ere ch rom atograp h ed a s p r e v io u s ly d e s c r i b e d . F or q u a n t i t a t i v e d e t e r m in a t io n s , t h e m ethod o f Lipm ann and T u t t l e (1 9 4 5 ) was e m p lo y ed . 65 I I I . EXPERIMENTAL RESULTS Q u a l i t a t i v e t r a p p in g e x p e r im e n ts w ere ru n b o th i n th e p r e s e n c e and a b s e n c e o f o x y g e n . The r e a c t i o n m ix tu r e in c lu d e d m agnesium s i n c e a t t h e tim e t h e s e e x p e r im e n ts w ere r u n t h e i n h i b i t o r y e f f e c t o f t h i s c a t i o n w as n o t s u s p e c t e d . The d a ta o b ta in e d (T a b le XIV) show t h a t an a c t i v e c a p r a te w as tr a p p e d b o th a e r o b i c a l l y and a n a e r o b i c a l l y . I t i s ap p a r e n t from th e r e s u l t s t h a t a c e t a t e a l s o i s a c t i v a t e d by th e enzyme p r e p a r a t io n . F o r t h e q u a n t i t a t i v e d e t e r m in a tio n s o f b i o l o g i c a l l y form ed h yd r oxam at e s , r e a c t i o n v e s s e l s w ere s e t up a s shown i n T a b le XV. D e te r m in a tio n s o f t h e h yd roxam ic d e r i v a t i v e s w ere c a r r ie d o u t on 1 ml a l i q u o t s o f t h e r e a c t i o n m ix tu r e s a f t e r 90 m in u te s in c u b a t io n ; t h e r em a in d er o f t h e r e a c t i o n m ix tu r e w as c o n c e n tr a t e d and ch ro m a to g ra p h ed . The g r e a t e s t amount o f h yd roxam ate fo r m a tio n o c c u r r e d i n t h e c o m p le te s y s te m , w h ile t h e am ounts form ed i n t h e s y s te m s o m it t in g ATP a n d /o r G O A w ere r e l a t i v e l y low (T a b le X V ). S in c e t h e S40 - 6 O f r a c t i o n em ployed was n e i t h e r d ia ly z e d n o r t r e a t e d w it h D o w e x -l-H C l, r e s i d u a l ATP and GoA c o u ld a c c o u n t f o r t h i s fo r m a tio n ( s e e T a b le V I I I ) . The R|. v a lu e s o b ta in e d i n th e q u a n t i t a t i v e t e s t s c o r r e sp o n d t o th e c o n t r o l v a lu e s f o r c a p r y l h y d ro x a m a te. T h ese d a ta e s t a b l i s h t h a t an a c t i v e c a p r a te i s 66 form ed a s th e f i r s t s t e p i n t h e o x id a t io n m ech an ism . TABUS XIV R f VALDES O P HYDROXYIAMim TRAPPED BIOLOGICAL ESTERS E x p e r i m ent N o. NHgOH S u b s t r a t e Rf. v a lu e s R^ v a lu e s o f com - obtained'*®* p a r a t iv e sta n d a r d s A c e t a t e C ap rate 1 100 ;iM 10 fiM c a p r a te 0 .9 7 100 ym 10 pM caprate'-"^ 0 .9 8 5 2 1 0 0 pm 10 pM c a p r a te 0 .9 6 0 .5 1 - 0 .5 4 0 .9 5 - 0 .9 8 100 pM 10 pM c a p r a te 0 .9 7 100 100 pM a c e t a t e 0 .5 3 100 100 pM a c e t a t e 0 .5 4 '-Enzyme c o n t r o l w ith o u t s u b s t r a t e w as n e g a t i v e , ^ ^ e a c t i o n ru n a e r o b i c a l l y . R e a c t io n m ix tu r e c o n ta in e d 0 . 2 m l S40 - 6 O f r a c t i o n ( 0 .1 5 mg N ), 120 u n i t s CoA, 108 jaM GSH, 1 3 .2 j k ATP, 2 jM and s u b s t r a t e and h y d ro x y la m in e a s i n d ic a t e d i n a t o t a l volum e o f 3 .0 m l. I n c u b a t io n 30® C, n it r o g e n atmos-* p h e r e . T r is b u f f e r , pH 7 . 4 . I n c u b a tio n was f o r 90 m in u te s . 67 TABIE XV QUANTITATIVE DETERMINATION O P BIOLOGICALLY FORMED GAPRYL G O A A d d it io n s t o r e - p.M h yd roxam ic R jg * v a lu e s o f v a lu e o f a c t i o n m ix tu r e a c id form ed^ a c t i v a t e d c o m p a r a tiv e d e r i v a t i v e sta n d a r d * * ATP, Mg GOA, GSH, 1 .0 ; 1 . 5 0 .9 7 ; 0 .9 7 ATP, Mg C oA , GSH, 2 .1 * * * 0 .9 6 GOA, GSH, M g 0 .5 - GSH, Mg 0 .3 - None 0 t o 0 .2 — 0 .9 5 - 0 .9 8 /^Lipmann and T u t t l e . ^ ^ B u t a n o l - a c e t i c a c id - w a t e r , 4 : 1 : 5 . * * * R e a c tio n ru n a e r o b i c a l l y . R e a c t io n m ix tu r e c o n ta in e d 1 . 0 m l S40 - 6O f r a c t i o n ( 0 .7 5 mg N ), 20 pM c a p r a te , 100 pM NHgOH, 2.pM Mg++, 5 pM KgHPO*, 8 2 pM GSH, and w h ere in d ic a t e d 200 u n i t s CoA, 1 5 .2 pM ATP, i n a t o t a l volu m e o f 5 . 0 m l. I n c u b a tio n 50® C f o r 90 m in u te s , n it r o g e n a tm o sp h e r e . T r is b u f f e r , pH 7 . 4 . CHAPTER VII THE OXIDATION O P ALIPHATIC PATTY ACIDS BY CELL-PREE SYSTEMS PROM_BACTERIA. DISCUSSION OP RESULTS Prom th e d a ta p r e s e n te d i n t h i s stu d y i t i s c o n c lu d e d t h a t t h e m echanism o f c a p r a te o x id a t io n by t h e o r g a n ism s em p loyed p a r a l l e l s th e m echanism o f f a t t y a c id o x id a t io n fo u n d i n mammalian s y s t e m s , and c o n s t i t u t e s a b e ta o x id a t io n o f an a c t i v a t e d com pound. T h is c o n c lu s io n i s s u b s t a n t i a t e d b y t h e f o l l o w i n g e x p e r im e n ta l e v id e n c e : ( 1 ) The d e m o n s tr a tio n o f a n ATP, CoA r e q u ir e m e n t f o r c a p r a te o x id a t io n by th e c e l l - f r e e s y s t e m s . ( 2 ) The tr a p p in g o f an a c t i v e e s t e r o f c a p r a te u n d er a n a e r o b ic c o n d i t i o n s . ( 3 ) The d e m o n s tr a tio n o f a c e t a t e fo r m a tio n from c a p r a te w it h th e c o n su m p tio n o f o n ly 1 pM o f o x y g en p e r m icro m o le o f c a p r a t e . The e s t a b lis h m e n t o f t h e r e q u ir e m e n t f o r ATP and CoA show s t h a t an a c t i v a t i o n o f th e f a t t y a c id s u b s t r a t e o c c u r s . T h is i s c o n fir m e d by tr a p p in g a c t i v e c a p r a te u n d er c o n d it io n s w here o x id a t io n c o u ld n o t o c c u r . D i r e c t demon s t r a t i o n t h a t t h e a c t i v a t e d compound i s c a p r y l CoA i s l a c k in g , h ow ever i t i s d i f f i c u l t to e x p la in th e d i s t i n c t r e q u ir e m e n t f o r CoA i f t h i s I s n o t th e c a s e . Prom t h e s e 69 i r e s u l t s , t h e r e f o r e , a c o n s i s t e a n a lo g y i s e s t a b l i s h e d b e tw e en t h e o r g a n ism s em p loyed ( S . m a r c e sc e n s and P . f l u o r e s - c e n s ) and c l . k lu y v e r i and a n im a l s y s te m s f o r t h e a c t i v a t i o n r e a c t i o n . The w ork o f s i l l i k e r and R it t e n b e r g (1 9 5 1 a , 1 9 5 1 b , and 1 9 5 2 ) and th e s t u d i e s w it h th e omega p h e n y l s u b s t i t u t e d f a t t y a c id s c o u ld n o t d i s t i n g u i s h b e tw e en a b e ta o x id a t io n m echan ism and a m u l t i p l e a l t e r n a t e o x id a t io n m ech an ism . C e l l - f r e e s t u d i e s w it h t h e sam e o rg a n ism s h o w ev er, c l e a r l y e s t a b l i s h e d t h e m echanism o f f a t t y a c id o x id a t io n by t h e s e o r g a n ism s a s b e in g a b e ta o x i d a t i o n . T h is c o n c lu s io n i s m o st s t r o n g l y d e m o n str a te d b y t h e d e t e c t i o n o f a c e t a t e a s a p r o d u c t o f c a p r a te o x id a t io n u s in g cru d e c e l l - f r e e s y s tem s o f P . f l u o r e s c e n s w h ic h consum e 1 pM o f o x y g e n p er m icro m o le o f s u b s t r a t e . I f m u l t i p l e a l t e r n a t e o x id a t io n w ere o c c u r r in g w it h th e e x t r a c t s o f t h i s o r g a n ism , no a c e t a t e w o u ld b e form ed a t t h i s l e v e l o f o x i d a t i o n . S i m i l a r l y , w it h S . m a r c e s c e n s , a c e t a t e w as form ed w it h t h e c o n su m p tio n o f 2 .5 pM o x y g en ^ e r m icro m o le s u b s t r a t e . In th e l a t t e r c a s e , a g a in , th e o x y g en u p ta k e i s i n s u f f i c i e n t t o form a c e t a t e b y th e th e o r y o f m u l t i p l e a l t e r n a t e o x i d a t i o n . E x p e r im e n ts e m p lo y in g a n im a l sy s te m s h ave shown a t h i o l y t i c c le a v a g e o f an a c t i v a t e d b e ta k e t o f a t t y a c id t o 70 y i e l d a c e t y l CoA and a f a t t y a c id fr a g m e n t, w it h tw o l e s s c a r b o n s , i n t h e a c t i v e fo r m . A lth o u g h t h e d a ta p r e s e n t e d show a b e ta o x id a t io n and c le a v a g e w it h t h e f o r m a t io n o f a c e t a t e , t h e y do n o t p r o v e t h a t a t h i o l y t i c c le a v a g e o c c u r s , and t h i s ca n o n ly be assum ed by a n a lo g y t o a n im a l s y s t e m s . Two p e c u l i a r i t i e s o f d a ta sh o u ld be m e n tio n e d . F i r s t , no ch r o m a to g r a p h ic e v id e n c e f o r th e e x i s t e n c e o f a c a p r y ly l CoA a s th e o th e r p r o d u c t o f c a p r y l CoA c le a v a g e by p . f l u o r e s c e n s was fo u n d . T h is an om aly may m e r e ly b e du e t o t h e i n a b i l i t y o f th e c h r o m a to g r a p h ic m ethod to d e t e c t th e q u a n t i t i e s p r e s e n t i n t h e r e a c t i o n m ix t u r e . S e c o n d ly , i f c a p r y ly l CoA i s t h e o th e r p r o d u c t, one m ust a c c o u n t f o r th e la c k o f i t s f u r t h e r o x id a t io n a s e v id e n c e d by th e o x y g en u p ta k e o f 1 pM o x y g e n p e r m icro m o le a c t i v e c a p r a t e . I t h a s b e e n shown t h a t c a p r y la t e i s o x id iz e d b y t h e s e p r e p a r a tio n s w it h th e c o n su m p tio n o f o n ly 1 pM o f o x y g en p er m icro m o le s u b s t r a t e (T a b le V I I ) . I f a t h i o l y t i c c le a v a g e o c c u r r e d d u r in g c a p r a te o x id a t io n , t h e e ig h t c a r bon fra g m en t s h o u ld h ave b e e n i n t h e a c t i v a t e d form t h e r e f o r e e li m in a t i n g a f u r t h e r r e q u ir e m e n t f o r ATP. Lack o f f u r t h e r o x i d a t i o n m ig h t be e x p la in e d by a l i m i t i n g q u a n t it y o f some c o f a c t o r ( s ) i n t h e h y d ro g e n t r a n s p o r t s y s te m . T h is e x p la n a t io n h o w ev er, i s n o t s a t i s f a c t o r y s i n c e d o u b lin g th e l e v e l o f s u b s t r a t e d o u b le s t h e o x y g e n c o n su m p tio n a t a ! c o n s t a n t l e v e l o f ATP and GoA. F u r th e r i n v e s t i g a t i o n o f t h i s e f f e c t i s r e q u ir e d b e f o r e a s u i t a b l e e x p la n a t io n ca n be o f f e r e d . The w ork o f S tad tm an and B a rk er (S ta d tm a n , 1 9 5 3 ) and M ahler (1 9 5 3 b ) d e m o n str a te d t h a t a c t i v a t e d d e r i v a t i v e s o f a lp h a b e ta u n s a t u r a t e d , b e ta h y d r o x y , and b e t a k e t o f a t t y a c id s a r e th e in t e r m e d ia t e s i n th e o x i d a t i o n . The o c c u r r e n c e o f t h e s e compounds h a s n o t b e e n d e m o n str a te d w it h th e p r e p a r a tio n s u s e d i n t h i s s t u d y . The p a r t i a l l y p u r i f i e d (S4 0 - 6 O f r a c t i o n ) enzyme p r e p a r a tio n from P . f l u o r e s c e n s h ow ever consum ed o n ly 0 .5 pM o f o x y g en p e r m icrom ole o f c a p r a t e . T h is i n d i c a t e s a b lo c k i n th e o x id a t io n b e f o r e th e b e t a k e t o c a p r a te s t a g e and i n d i c a t e s t h a t some l e s s o x id iz e d p r o d u c t o f c a p r a te o x id a t io n a c c u m u la te s . C h rom atograp h ic and c h e m ic a l t e s t s on th e r e a c t i o n m ix tu r e f a i l e d t o show a c e t a t e o f k e to g r o u p s b u t d id r e v e a l an un i d e n t i f i e d s p o t w it h a n R f o f 0 .2 2 a s an end p r o d u c t. T h is s p o t h a s n o t b e e n i d e n t i f i e d y e t due t o th e la c k o f a v a i l a b le s ta n d a r d s ; c o n s e q u e n t ly , t h e r e i s no b a s i s a s y e t f o r d e c id in g b e tw e en an a lp h a b e ta u n s a tu r a te d or b e t a h y d ro x y a c t i v e c a p r a te a s th e p r o d u c t, or p erh a p s an e n t i r e l y d i f f e r e n t com pound. From th e e x p e r im e n ta l d a ta o b ta in e d th e m echanism o f f a t t y a c id o x id a t io n b y t h e b a c t e r i a em p loyed can be j I 72 o u t l i n e d a s sum m arized i n F ig u r e 7 . I t i s a b e ta o x i d a t i o n j d i f f e r i n g from t h a t p r o p o se d by Knopp and e la b o r a t e d by D a k in (1 9 0 9 ) i n t h a t a CoA a c t i v a t e d compound u n d e r g o e s o x id a t io n r a t h e r th a n t h e f r e e f a t t y a c id a s s u c h . I t i s a n a lo g o u s i n d e t a i l t o m echanism s, a lr e a d y p r e s e n t e d by B ark er (S ta d tm a n , 1 9 5 3 ) f o r t h e o x id a t io n o f lo w e r f a t t y a c id s b y Cl* k l u y v e r i , and by num erous w o r k e rs a lr e a d y c i t e d f o r o x id a t io n o f f a t t y a c id s b y a n im a ls . The a c t i v a t i o n o f t h e s u b s t r a t e and th e s p l i t t i n g o f f o f a tw o - c a r b o n fra g m e n t h a s b e e n c o n c l u s i v e l y sh ow n . The i n d ic a t e d in t e r m e d ia t e s t e p s . s t i l l r e q u ir e f u r t h e r v e r i f i c a t i o n . g o o g" o w* g o 0 0 44 1 iH iH 0 0 <d O 44 o 0 'b o o o 0 O o 0 u o o od t5 r f — g 0 - » o " g «H W m 44 o o Ü C i 0 a s W M U o O A 0 3 03 § O 0-0 § m § o O o 0=0 w o « « % 43 I s Q ) < D ■ f i ■3 O 0 1 o 0 o O 0 o o O o o O 0 C i o w W 0 o . O . M 0 %-^11 O w w w o O o - O C i C i « W M W o O o 03 03 03 o o o ' w * 8=0 {3? O § o 1 3 ? O 8 § o o 4 4 c d 44 -►•H U _ 5 § 44 .H 4 4 T f * H O O M E4 E4 o ^ l § 4 4 »H O « & 4 O H o O ___ t ? o g f4 4 4 ■ I g H i < D 0 44 0 E4 O O 0 o o rH O o rH o o 0 ci 0 O tu o 03 hû et 0 ----- ^ •H o O 44 0 0 0 03 03 0 O o 03 03 03 o îS § - g § « o O ü •H o 44 o —— > as 0 »0 03 •H O g O <jj + O 0 o O <3 O 0 » 0 43 03 « O ^ O 0 = 0 o P 0 a 44 03 03 o O O P3 03 E4 P Q 44 c d "0 S o o g •H 44 O c d f i 0 (O 1 * O H - s H < « * C / 3 44 « % Q d 44 O € d ^ *0 O < | > rH M Xi o o 4 4 73 iH n O g 44 44 O K c d 0 •P 0 g 1 0 O ts O *H *0 (O A • rH A < d A < | C Q | I col CHAPTER V III RESUME The m echanism o f f a t t y a c id o x id a t io n by b a c t e r i a w as i n v e s t i g a t e d b y u s in g th e te c h n iq u e o f s im u lta n e o u s a d a p t a t io n and b y s tu d y in g c e l l - f r e e e x t r a c t s * I t was shown t h a t a v a r i e t y o f b a c t e r i a i s a b le t o p a r t i a l l y o r c o m p le te ly o x i d i z e c e r t a i n p h e n y l s u b s t i t u t e d f a t t y a c id s * On th e b a s i s o f a d a p t a t io n p a t t e r n s and o x y g e n u p ta k e d a ta i t h a s b e e n d e m o n str a te d ( 1 ) t h a t shortei» c h a in p h e n y l f a t t y a c id s a s su c h a r e n o t d i r e c t i n t e r m e d ia te s in t h e o x id a t io n o f lo n g e r c h a in p h e n y l su b s t i t u t e d f a t t y a c id s and ( 2 ) t h a t n e v e r t h e l e s s , a b e ta o x i d a t i o n o f th e lo n g e r c h a in compounds o c c u r s* The d a ta i n d i c a t e t h a t th e o x i d a t i o n o f lo n g c h a in p h e n y l f a t t y a c id s in v o lv e s an a c t i v a t i o n f o llo w e d by a b e t a o x id a t io n o f th e a c t i v a t e d compounds w h ic h i n some o r g a n ism s p r o c e e d s t o t h e b e n z o a te or p h e n y la c e t a t e l e v e l * The o x id a t io n o f th e lo w e r p h e n y l f a t t y a c id s (p h e n y lb u ty r a te t o b e n z o a te ) i n f o u r o f t h e o rg a n ism s t e s t e d a p p a r e n tly p r o c e e d s v i a t h e a r o m a tic schem e d e s c r ib e d b y o th e r s * H ow ever, th e d a ta s u g g e s t t h a t an a l t e r n a t e pathw ay p r o c e e d in g d i r e c t l y from th e a c t i v a t e d p h e n y la c e t a t e and b e n z o a te a l s o e x i s t s i n s o i l i s o l a t e S . 61* 75 C e l l - f r e e e x t r a c t s w e re p r e p a r e d from S . m a r c e s c e n s and ?* f l u o r e s c e n s b y g r in d in g w ith f i n e g l a s s p ow der. The c r u d e e x t r a c t s o f S . m a r c e sc e n s o x id iz e d c a p r a t e and c a p r y la t e t o th e e x t e n t o f 2 .5 yM o f o x y g en p e r m ic r o m o le o f s u b s t r a t e i n th e p r e s e n c e o f a su p p lem en t m ix tu r e c o n t a in in g ATP, CoA, GSH, and Mg. I f ATP w ere o m itte d , t h e ox y g en u p ta k e w as m a rk ed ly r e d u c e d . The a d d it io n o f cy to ch ro m e c , DPN, s u c c i n a t e , r i b o f la v in e - 5 - p h o s p h a t e t o t h e b a s ic su p p lem en t d id n o t in c r e a s e t h e o x y g e n c o n su m p tio n , C h rom atograp h ic a n a l y s i s o f th e r e a c t i o n sy s te m i n d ic a t e d th e p r e s e n c e o f a c e t a t e ( a c e t y l CoA) and an u n i d e n t i f i e d s p o t , p r o b a b ly a fo u r carb on r e s id u e o f c a p r y la t e a t th e o x id a t io n l e v e l o f b e ta h y d ro x y b u t y r a t e . Crude e x t r a c t s fro m P . f l u o r e s c e n s o x id iz e d c a p r a te i n th e p r e s e n c e o f ATP, CoA, GSH, and Mg w ith an u p ta k e o f 1 o f o x y g e n p e r m icro m o le o f s u b s t r a t e . As w it h S e r r a t i a , th e o x id a t io n c o u ld n o t be in c r e a s e d by th e ad d i t i o n o f v a r io u s c o f a c t o r s . S t u d ie s w ith D o w ex -1 - h y d r o c h lo r id e t r e a t e d and n o n - t r e a t e d c e l l - f r e e e x t r a c t s e s t a b l i s h e d t h a t ATP and CoA w ere e s s e n t i a l c o f a c t o r s f o r t h e o x id a t io n o f c a p r a te and in d ic a t e d t h a t a c t i v a t i o n o f th e f a t t y a c id s w as an e s s e n t i a l p r e r e q u i s i t e t o th e o x i d a t i o n , C o n c lu s iv e e v id e n c e f o r th e a c t i v a t i o n s t e p was 76 a f f o r d e d by th e t r a p p in g o f a c t i v e c a p r a te u n d er a n a e r o b ic c o n d it io n s and i t s d e m o n s tr a tio n by q u a l i t a t i v e and q u a n t i t a t i v e m e th o d s. A c e t a t e , w h ic h was n o t o x id iz e d by t h i s p r e p a r a t io n , was d e t e c t e d c h r o m a to g r a p h ic a lly as an end p r o d u c t o f c a p r a te o x i d a t i o n . The a c c u m u la tio n o f a c e t a t e w it h th e u t i l i z a t i o n of^ o n ly 1 jM o f o x y g en p e r m icr o m o le s u b s t r a t e e s t a b l i s h e s a b e t a o x i d a t i o n m echanism and e li m i n a t e s m u l t i p l e a l t e r n a t e o x id a t io n a s a p o s s i b i l i t y . P a r t i a l p u r i f i c a t i o n o f t h e cru d e c e l l - f r e e e x t r a c t o f p . f l u o r é s c e n s was a c c o m p lis h e d . by ammoniiam s u l f a t e f r a c t i o n a t i o n . The f a t t y a c id o x id a s e s y s te m w as o b ta in e d i n t h e 4 0 -6 0 p e r c e n t s a t u r a t e d f r a c t i o n . The p r e p a r a t io n consum ed o n ly 0 ,5 jüM o f o x y g en p e r m icro m o le o f c a p r a t e . C h ro m a to g ra p h ic and c h e m ic a l t e s t s show ed th e a b s e n c e o f a c e t a t e and k e t o a c id s b u t r e v e a le d an a s y e t u n i d e n t i f i e d compound (R^ 0 ,2 2 ) a s an end p r o d u c t. The a d d i t io n o f f l u o r i d e t o th e r e a c t i o n m ix tu r e s t im u la t e d c a p r a te o x id a t io n b y t h e cru d e enzym e s y s te m . The a d d i t io n o f s m a ll am ounts o f m agnesium t o th e r e a c t i o n m ix tu r e g r e a t l y l i i h i b i t e d c a p r a te o x id a t io n by a d ia ly z e d S40 - 6 O p r e p a r a t io n . I t was c o n c lu d e d t h a t i f a m agnesium r e q u ire m en t e x i s t s f o r th e a c t i v a t i o n a n d /o r o x i d a t i o n , i t i s m et b y t r a c e s o f t h i s d i v a l e n t i o n . 77 From th e e v id e n c e o b ta in e d i t i s c o n c lu d e d t h a t t h e m echanism o f f a t t y a c id o x id a t io n by th e o r g a n ism s em ployed p a r a l l e l s th e m echanism w h ic h i s fo u n d i n m am m alian s y s te m s , T h is m echanism i s a b e t a o x id a t io n o f a n ATP-CoA a c t i v a t e d f a t t y a c id w ith o u t t h e fo r m a tio n o f f r e e f a t t y a c id in t e r m e d ia t e s d u r in g t h e o x i d a t i o n . BIBLIOGRAPHY BIBLIOGRAPHY B a r k e r, H* A . 1937 The p r o d u c tio n o f c a p r o ic and b u t y r ic a c id s by th e m eth an e fe r m e n t a t io n o f e t h y l a l c o h o l . A r c h , M ik r o b io l,, 8 , 4 1 6 - 4 2 1 , B a r k e r , H, A . , Kamen, M, D ., and B o m s t e l n , B , T . 1945 The s y n t h e s i s o f b u t y r ic and c a p r o ic a c id s from e th a n o l and a c e t i c a c id by C lo s t r id iu m k l u y v e r i , P r o c , N a t l, A c a d , S c i , Ü , S , , 3 1 , 3 7 3 - 5 8 1 , B e in e r t , H, and S t a n s l y , P , G, 1 9 5 3 A sym m etric l a b e l i n g o f a c e t o a c e t a t e b y e n z y m a tic a c e t y l e x c h a n g e w it h a c e t y l coenzym e A , J , B i o l , Chem ,, 2 0 4 , 6 7 - 7 6 , B o rn st e i n , B , T , and B a r k e r , H, A , 1948a The e n e r g y m e ta b o lis m o f C lo s tr id iu m k lu y v e r i and t h e s y n t h e s i s o f f a t t y a c i d s , J , B i o l , Chem ,, 1 7 2 , 6 5 9 , B o r n st e i n , B , T , and B a r k e r , H, A , 1948b The n u t r i t i o n o f C lb s tr id iu m k l u y v e r i , J , B a c t ,, 5 5 , 2 2 3 -2 3 0 , B r e u sc h , F , L , 1948 The b io c h e m is t r y o f f a t t y a c id c a t a b o lis m , A d v, i n E n zym e,, 8 , 3 4 3 -4 2 3 , C h e ld e lin , V, H, and B e in e r t , H, 1 9 5 2 S t u d ie s on t h e c y c lo p h o r a s e s y s te m , XXV, F a t t y a c i d o x i d a t i o n i n t h e r a b b it l i v e r s y s te m , B io c h e m ic a e t B io p h y s ic a A c t a , 9 , 6 6 1 -6 7 3 , D a k in , H, D , 1 9 0 9 The mode o f o x id a t io n i n t h e a n im a l o rg a n ism o f p h e n y l d e r i v a t i v e s o f f a t t y a c i d s , V I, J , B i o l , C hem ,, 6 , 2 3 5 -2 4 3 , D ix o n , M, 1 953 A nomogram f o r ammonium s u lp h a t e s o l u t i o n s . B io chem , J , , 4 5 7 - 4 5 8 , ^ E v a n s, W, C, 1947 O x id a tio n o f p h e n o l and b e n z o ic a c id by some s o i l b a c t e r i a . B loch em , J , , 4 1 , 3 7 3 -3 8 2 , G r a f f l i n , A , L , and G r e e n e , D . E , 1948 S t u d ie s on t h e c y c lo p h o r a s e s y s te m , I I , The c o m p le te o x id a t io n o f f a t t y a c i d s , J . B i o l , C hem ,, 1 7 6 , 9 5 -1 1 5 , G reen , D , E , 1948 The c y c lo p h o r a s e s y s t e m . The R ecord o f C h em ical P r o g r e s s , S p r in g I s s u e , 7 - 1 3 , 80 G reen , D# E . , G oldm an, S . , M il, S . and B e in e r t , H, 1 9 5 5 a The a c e t o a c e t a t e a c t i v a t i o n and c le a v a g e enzyme s y s te m , J , B i o l , Chem ., 2 0 2 , 1 5 7 -1 5 0 . : G reen , D . E , and M Ü , S . 1953b P a t t y a c id o x id a t io n w it h s o l u b l e enzym es from a n im a l t i s s u e s . F e d . P r o c ,, 1 2 , 211. J o w e tt , M, and Q u a s t e l, J , H, 1935 S t u d ie s on f a t m eta b o lis m , I I I , The fo r m a tio n and breakdow n o f a c e t o - a c e t i c a c id i n a n im a l t i s s u e s , B ioch em , J , , 2 9 , 2 1 8 1 - 2 1 9 1 , K ennedy, E . P , and B a r k e r , H, A , 1 9 5 1 B u ty r a te o x id a t io n i n t h e a b s e n c e o f in o r g a n ic p h o sp h a te by C lo s t r id iu m k l u y v e r i . J . B i o l , Chem ., 1 9 1 , 4 1 9 - 4 3 8 , K en n ed y, E , P , and L e h n in g e r , A . L , 1 9 5 2 The e n z y m a tic o x i d a t i o n o f f a t t y a c i d s . In P h osp h oru s M e ta b o lism , V o l, 1 , E d it e d b y M cE lroy, W. b . , and G la s s , B , The Johns Hopkins Press, Baltimore, Md, K hoop, P , 1904 D er abban a r o m a tis c h e r f e t t sa u r e n im Tierkorper, Beit, Z , Chem, Phy, H. Path,, £ , 1 5 0 -1 6 2 . K o rn b erg , A , and P r i c e r , W, E ., J r . 1953 E n zy m a tic s y n t h e s i s o f t h e coen zym e A d e r i v a t i v e s o f lo n g c h a in f a t t y a c i d s , J , B i o l , c h e m ,, 2 0 4 , 3 2 9 -3 4 3 , L ard y, H. A . 1 9 5 2 A t h e o r y c o n c e r n in g th e m echanism o f f a t t y a c id o x id a t io n and s y n t h e s i s and o f carb on d io x id e f i x a t i o n , P r o c . H at. A c a d . S c i , , 3 8 , 1 0 0 3 - 1 0 1 3 . L e h n in g e r , A . L . 1945 On th e a c t i v a t i o n o f f a t t y a c id o x i d a t i o n , J , B i o l , Chem ., 1 6 1 , 4 3 7 - 4 5 1 , L e h n in g e r , A , L , 1950 The e n z y m a tic o x i d a t i o n o f f a t t y a c i d s . The R ecord o f C h em ica l P r o g r e s s , S p r in g I s s u e , 7 5 - 8 2 , L e h n in g e r , A , L . 1952 E nzym ic o x id a t io n o f f a t t y a c i d s . B io c h e m ic a l s o c i e t y S y m p o sia , H o. 6 6 - 7 9 . Lipm ann, P , 1950 B io s y n t h e t ic m ec h a n ism s. The H arvey | L e c tu r e S e r i e s , S e r . 4 4 , 1 9 4 8 -1 9 4 9 , 9 9 - 1 2 3 , 81 Lipm ann, P . 195 3 On c h e m is tr y and f u n c t i o n o f coenzym e A . B a c t. R e v s ., 1 7 , 1 - 1 6 . Lipm ann, P . , J o n e s , M. E . , B la c k , S . and F ly n n , R . M. 1 953 The m echanism o f th e A T P -G o A -a ceta te r e a c t i o n . J . C e l l , and com p. P h y s i o l * , 4 1 , S u p p . 1 , 1 0 9 -1 1 2 . Lipm ann, P . and T u t t l e , 0 . L . 1945 D e te r m in a tio n o f a c y l p h o sp h a te s* J* B i o l . C hem ., 1 5 9 , 2 1 - 2 8 . Lipm ann, P . and T u t t l e , . C. L . 1 950 L ip a s e - c a t a ly z e d c o n d e n s a t io n o f f a t t y a c id s w it h h y d ro x y la m in e . B io ch em ica e t B io p h y s ic a A c t a , ^ 3 0 1 -3 0 9 . L ugg, J . W. H. and O v e r a ll, B . T . 1948 *^0ne** and " tw o - d im e n sio n a l'* p a r t i t i o n ch r o m a to g r a p h ic s e p a r a t io n s o f o r g a n ic a c id s on an i n e r t s h e e t s u p p o r t . A u s t r a lia n J . S c i . R e s ., 1 , 9 8 - 1 1 1 , L ynen, P , and O choa, S . 1 9 5 3 Enzym es o f f a t t y a c id m eta b o l is m , B io c h e m ic a e t B io p h y s ic a A c ta , 1 2 , 2 9 9 -3 1 4 , L yn en , P . , R e ic h e r t , E , and R u e f f , L . 1 951 Zum b i o l o g i s - | ch en Abbau d e r E s s i g s a u r e . V I . " A k t iv ie r t e E s s ig s & u r e ," ih r e I s o li e r u n g a u s H efe und ih r e ch era isch e H a tu r, A n n ., 5 7 4 , 1 7 9 - 1 8 9 , L yn en , P . , Wes s e l y , L ., W e ila n d , 0 , and R u e f f , L . 1 9 5 2 R o le o f coenzym e A i n f a t t y a c id m e ta b o lis m . Z t s c h r . A ngew , C hem ., 6 4 , 6 8 7 , C ite d by M ah ler, R, R, 1953 F e d , P r o c ,, 1 2 ~ 6 9 4 - 7 0 2 . M a h ler, H. R . 1953a B u ty r y l CoA d e h y d r o g e n a s e , a c u p r o - f l a v o - p r o t e i n , J , Am. Chem. S o c . , 7 5 , 3 2 8 8 -3 2 8 9 , M ah ler, H. R . 1953b R o le o f coenzym e A i n f a t t y a c id m e ta b o lis m . F e d . P r o c ., 1 2 , 6 9 4 - 7 0 2 . M a h ler, H. R ., W a k il, S . J . and B ock , R . M. 1 953 S t u d ie s on f a t t y a c id o x i d a t i o n . I , E n zy m a tic a c t i v a t i o n o f f a t t y a c i d s , J , B i o l , Chem ,, 2 0 4 , 4 5 3 -4 6 8 , Munoz, J . M. and L e L o ir , L , P , 1943 P a t t y a c id o x i d a t i o n by l i v e r en z y m e s, _ J , B i o l , c h e m ., 1 4 7 , 3 5 5 - 3 6 2 . N o v e l l i , G, D . 1 9 5 3 S t u d ie s c o n c e r n in g th e s t r u c t u r e o f coenzym e A . J . C e l l , and Comp, P h y s i o l , , 4 1 , Supp, 1 , 6 7 - 8 8 . 82 S i l l i k e r , J . H. 1950 S t u d ie s on th e a e r o b ic o x id a t io n o f f a t t y a c id s by b a c t e r i a . 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The e f f e c t o f 2 ,4 - d i n it r o p h e n o l on t h e o x i d a t i o n o f f a t t y a c id s by S e r r a t i a m a r c e s c e n s . J . B a c t ,, 6 4 , 1 9 7 - 2 0 5 . S t a d i a , W. C, 1945 The in te r m e d ia r y m e ta b o lism o f f a t t y a c i d s . P h y s i o l , R e v ., 2 5 , 3 9 5 -4 4 1 , S ta d tm a n , B , R , 1 9 5 3 D i s c u s s i o n , M e ta b o lic r e l a t i o n s o f coenzym e A . F e d . P r o c ., 1 2 , 6 9 2 -6 9 3 . S ta d tm a n , E . R . and B a r k e r , H, A . 1949a B a t t y a c id s y n t h e s i s b y enzym e p r e p a r a tio n s , o f C lo s tr id iu m k lu y v e r i, I , P r e p a r a tio n o f c e l l - f r e e e x t r a c t s t h a t c a t a l y z e th e c o n v e r s io n o f e t h a n o l and a c e t a t e t o b u ty r a te and c a p r o a te . J , B i o l , € h e m ,, 1 8 0 , 1 0 8 5 -1 0 9 3 . S ta d tm a n , E . R . and B a r k e r , H. A . 1949b P a t t y a c id s y n t h e s i s by enzym e p r e p a r a tio n s o f C lo s t r id iu m k l u y v e r i , 7 . A c o n s id e r a t io n o f p o s t u l a t e d f o u r - ca rb o n in t e r m e d ia t e s i n b u t y r a t e s y n t h e s i s , J , B i o l , Chem ,, 1 8 1 , 2 2 1 -2 3 5 , S ta d tm a n , E . R , and B a r k e r , H, A . 1 950 F a t t y a c id s y n t h e s i s by enzyme p r e p a r a tio n s o f C lo s t r id iu m k l u y v e r i . V I. R e a c t io n s o f a c y l p h o s p h a te s . J . S i o l . ChemV, 184, 7 6 9 - 7 9 3 , ------ S ta d tm a n , E . R . and H o v e l l i , 0 . D* and Lipm ann, P . 1951 Coenzyme A f u n c t i o n i n and a c e t y l t r a n s f e r by th e p h o s p h o t r a n s a c e t y la s e s y s te m . J . B i o l , Chem,, 1 9 1 , 3 6 5 - 3 7 6 .---------------------------------------------------------------------------------- r 8 3 S t a n i e r , R . Y . 1 9 6 0 P rob lem s o f b a c t e r i a l o x i d a t i v e m e ta b o lis m , B a c t , R e v s ,, 1 4 , 1 7 9 -1 9 1 , S t e r n , J , R ,, Coon, M, J , , and D e l C a m p illo , A . 1953 E n zy m a tic breakdow n and s y n t h e s i s o f a c e t o a c e t a t e . N a tu r e , 3 ^ , 2 8 - 3 0 , U m b r e it, W, W ,, B u r r is , R , H, and S t a u f f e r , J . P , 1949 M anom etric t e c h n iq u e s and t i s s u e m e ta b o lis m , B u r g e ss P u b l, CO., M in n e a p o lis , M inn, V erk a d e, P . E . and van d e r L e e , J , 1 9 3 2 P r o c , K o n in k l, Akad* We t e n s c h a p , A m sterd a m ,, 3 5 , 2 5 1 . C it e d by B r e u sc h , P , L , 1 9 4 8 , W arburg, 0 , and C h r i s t i a n , W, 1941 G h em isch er M echanism us d e r Pluorid-H em m ung d er. O aru ng, N a t u r w is s e n s c h a fte n , 2 9 , 5 9 0 . W ein h o u se, S . 1 9 5 2 The s t r u c t u r e o f " a c t iv e a c e t y l " and a th e o r y o f f a t t y a c id c a t a b o lis m . A r c h , B io ch em , and B io p h y s ic s , 2 3 9 -2 4 1 , W o lfe , J , B . 1954 M eta b o lism o f m a lo n a te . b y Pseudom onas f l u o r e s c e n s , D i s s e r t a t i o n , U n iv e r s i t y o f S o u th e r n California, U n iverM fv o t O sllio fB iB UMI Number: DP23897 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. BtessftaÈion RuhlisNng UMI DP23897 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346

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Creator Ivler, Daniel (author)

Core Title Studies on the bacterial oxidation of saturated fatty acids and their derivatives

School Graduate School

Degree Doctor of Philosophy

Degree Program Bacteriology

Degree Conferral Date 1954-06

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biological sciences,OAI-PMH Harvest

Language English

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c30-262619

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Legacy Identifier DP23897.pdf

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Document Type Dissertation

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Type texts

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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