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Reaction of thiol compounds with pyridine nucleotides

Reaction of thiol compounds with pyridine nucleotides

VOL. 23 (z957) PRELIMINARY NOTES 221 Reaction of thiol compounds with pyridine nucleotides I t h a s p r e v i o u s l y been r e p o r t e d t h a...

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VOL. 23 (z957)

PRELIMINARY NOTES

221

Reaction of thiol compounds with pyridine nucleotides I t h a s p r e v i o u s l y been r e p o r t e d t h a t a c o m p l e x is f o r m e d b e t w e e n D P N a n d h y d r o x y l a m i n e in t h e presence of horse liver alcohol d e h y d r o g e n a s e 1. A similar c o m p l e x h a s also been o b s e r v e d b e t w e e n h y d r o x y l a m i n e , t h e p y r i d i n e - 3 - a l d e h y d e a n a l o g u e of D P N a n d y e a s t alcohol d e h y d r o g e n a s e ~. R e c e n t l y , TERAYAMA AND V E S T L I N G 3 h a v e reported a c o m p l e x b e t w e e n liver lactic d e h y d r o g e n a s e , D P N a n d s o d i u m sulfide. W e wish to p r e s e n t evidence, i n d i c a t i n g t h a t c o m p l e x f o r m a t i o n b e t w e e n p y r i d i n e c o e n z y m e , e n z y m e , a n d a nucleopbilic c o m p o u n d s t r u c t u r a l l y related to t h e s n b s t r a t e , is a general r e a c t i o n of all d e h y d r o g e n a s e s w h i c h c a r r y o u t t h e following t y p e of reaction: R1

V

H-C-0H

R1 +

DPN

r

>

I

C~0

+

DPNH

+

H+

i

R2

R2

W e h a v e f o u n d t h a t t h i s t y p e of d e h y d r o g e n a s e will i n t e r a c t w i t h thiol derivatives a n d p y r i d i n e nucleotides. All thiols e x a m i n e d , i n c l u d i n g s o d i u m sulfide, m e r c a p t o a c e t i c acid, m e r c a p t o s u c c i n i c acid, t h i o e t h a n o l a n d n - a l k y l - m e r c a p t a n s , react n o n - e n z y m i c a l l y w i t h pyridine nucleotides to form a d d i t i o n p r o d u c t s . I n s o m e i n s t a n c e s w h e r e D P N s h o w s little reaction, D P N a n a l o g u e s (i.e. t h e a c e t y l p y r i d i n e or p y r i d i n e - 3 - a l d e h y d e a n a l o g u e s of D P N 4) will f o r m a d d i t i o n complexes. I n t h e presence of a suitable d e h y d r o g e n a s e this reaction b e t w e e n c o e n z y m e a n d thiol c o m p o u n d b e c o m e s TABLE I THE ENZYME-COENZYME-SUBSTRATE COMPLEX FOR DIFFERENT DEHYDROGENASEG S p e c t r a were t a k e n in o.I M p h o s p h a t e buffer, p H 7.4. T h e reference cell c o n t a i n e d t h e thiol c o m p o u n d , t h e e x p e r i m e n t a l cell thiol d e r i v a t i v e a n d e n z y m e . A f t e r this s p e c t r u m w a s t a k e n , t h e c o e n z y m e w a s a d d e d to b o t h reference cell a n d e x p e r i m e n t a l cell. T h e a d d i t i o n - c o m p l e x s p e c t r u m was c o m p u t e d from t h e difference of t h e t w o s p e c t r a obtained. A B e c k m a n s p e c t r o p h o t o m e t e r , m o d e l DE: was used t h r o u g h o u t .

Dehydrogenase

L i v e r alcohol dehydrogenase Y e a s t alcohol dehydrogenase Beef h e a r t lactic dehydrogenase Skeletal muscle lactic dehydrogenase Liver lactic dehydrogenase Pig h e a r t malic dehydrogenase Beef liver g l u t a m i c acid d e h y d r o g e n a s e R a b b i t m u s c l e glycerol phosphate dehydrogenase

Coenzyme or eoenzyme analogue

Thiol-derivative

Emax otzymic complex

Ema% no~-enzymic complex

mrs

mrs

DPN

ethylmercaptan

315

33 °

APDPN*

ethylmercaptan

34 °

355

DPN

mercaptosuccinic acid mercaptosuccinic acid s o d i u m sulfide

31o

33 °

31o

33 °

32o**

335

mercaptosuccinic acid mercaptoacetic acid p r o p a n e - 1,2dithiol

320

355

315

330

345 * **

355

DPN DPN Py-3A1DPN * D PN Py-3AIDPN*

* A P D P N a n d P y - 3 A 1 D P N s t a n d for t h e a c e t y l p y r i d i n e a n d p y r i d i n e - 3 - a l d e h y d e a n a l o g u e s of D P N . ** D a t a t a k e n from TERAYAMA AND V E S T L I N G 8. *** I n c o n t r a s t w i t h all o t h e r c o m p l e x e s reported in this table, t h e c o m p l e x on this e n z y m e f o r m s with a m e a s u r a b l e rate, r a t h e r t h a n i n s t a n t a n e o u s . § C o n t r i b u t i o n No. 151 of t h e M c C o l l u m - P r a t t I n s t i t u t e . S u p p o r t e d in p a r t s b y g r a n t s from t h e A m e r i c a n Cancer Society as r e c o m m e n d e d b y t h e C o m m i t t e e on G r o w t h of t h e N a t i o n a l R e s e a r c h Council, a n d t h e N a t i o n a l Cancer I n s t i t u t e of t h e N a t i o n a l I n s t i t u t e s of H e a l t h (Grant No. 2347-C).

222

PRELIMINARY NOTES

VOL. 9.3 (1957)

m u c h more favorable. F o r example, in the case of yeast alcohol dehydrogenase the reaction between the acetylpyridine analogue of D P N and e t h y l m e r c a p t a n is a p p r o x i m a t e l y a million times more favorable t h a n w h e n the reaction is carried out in the absence of enzyme. I t is of interest to note t h a t a l t h o u g h D P N shows little reaction with y e a s t alcohol dehydrogenase and e t h y l m e r c a p t a n , crystalline horse liver alcohol dehydrogenase will readily form a complex with the thiol c o m p o u n d and D P N (see Table I). The reaction between the enzyme, coenzyme and s u b s t r a t e analogue exhibits specificity. Only those thiol derivatives which s h o w a sufficient s t r u c t u r a l resemblance to the n o r m a l s u b s t r a t e of the various dehydrogenases will react with the pyridine nucleotides. F o r example, beef heart lactic dehydrogenase will react with mercaptoacetic or mercaptosuccinic acids, b u t not with 2mercaptoethanol. Similar specificity is s h o w n by skeletal muscle lactic dehydrogenase. The alcohol dehydrogenases will only form complexes with the n-alkyl mercaptans. Malic dehydrogenase will form a complex with mercaptosuccinic acid b u t n o t with e t h y l m e r c a p t a n . This enzyme a p p e a r s to give a far more favorable complex with pyridine-3-aldehyde analogue of D P N t h a n with D P N . Crystalline beef liver glutamic dehydrogenase forms a complex with D P N and mercapto-acetic acid, b u t n o t with mercaptosuccinic acid. Crystalline r a b b i t muscle glycerophosphate dehydrogenase shows a complex with propane-i,2-dithiol b u t not with thioethanol. Again the complex with the pyridine-3-aldehyde analogue a p p e a r s to be far more favorable t h a n with DPN. The chemical reaction between h y d r o x y l a m i n e and D P N has a m a x i m u m at 315 m#5; in the presence of the horse liver alcohol dehydrogenase the m a x i m u m is shifted to 300 m # 1. I n a similar m a n n e r , the a b s o r p t i o n m a x i m a of the enzymically-bound thiol complexes are shifted toward s h o r t e r wavelengths (Table I). I n all cases, where an enzymic reaction has been observed, a non-enzymic complex has also been detected. The results of this work strongly s u p p o r t the previously proposed hypothesis t h a t an addition reaction of s u b s t r a t e to coenzyme m a y be the first step in the reaction catalysed b y this class of dehydrogenase s. Details of the kinetic implications and properties of the thiol-pyridine nucleotide complexes will be published shortly.

~l/IcCollum Pratt Institute, The Johns Hopkins University, Baltimore, Md. (U.S.A.)

JAN VAN EYS NATHAN O. KAPLAN FRANCIS E. STOLZENBACH

1 N. O. KAPLAN AND M. M. CIOTTI, J. Biol. Chem., 211 (1954) 431. J. VAN EYs, IV[. M. CIOTTI AND N. O. KAPLAN, Biochim. Biophys. Acta, forthcoming paper. 3 H. TERAYAMA AND C. S. VESTLING, Biochim. Biophys..4eta, 2o (1956) 586. 4 N. O. KAPLAN AND M. IV[. CIOTTI, J. Biol. Chem., 22i (1956) 823. 5 R. M. BURTON AND N. O. KAPLAN, J. Biol. Chem., 211 (1954) 447. Received October I5th, 1956

The enzymic oxidation of reduced vitamin K 3 (menadione) A l t h o u g h enzymes catalysing the reduction of v i t a m i n K a (menadione) z or of v i t a m i n K 1 2 by reduced d i p h o s p h o p y r i d i n e nucleotide ( D P N H ) are known, there are no reports in the literature t h a t the r e s u l t a n t h y d r o q u i n o n e s can be re-oxidized b y mitochondrial preparations. B o t h h y d r o q u i n o n e s are, in fact, rapidly auto-oxidizable b y air at n e u t r a l pH, b u t at p H 6.2 4, in the presence of e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA), the auto-oxidation of the h y d r o q u i n o n e of menadione (K3H2) is very slow. I t has n o w been shown, b y working at this pH, t h a t sarcosomal (mitochondrial) f r a g m e n t s prepared from horse h e a r t (Keilin and H a r t r e e heart-muscle preparation) actively catalyse the oxidation of KaH ~ in the absence of added cytochrome c. The special interest of this reaction lies in the fact that, whereas a large n u m b e r of substances (such as ascorbic acid, benzohydroquinone) can be oxidized b y this p r e p a r a t i o n in the presence of added cytochrome c3, only succinate, D P N H and p-phenylenediamine are oxidized in the absence of the added cytochrome. The oxidation of K3H 2 resembles t h a t of succinate and D P N H r a t h e r t h a n t h a t of p-phenylenediamine, since it is completely inhibited b y a n t i m y c i n A. The K3H,2 oxidase activity was m e a s u r e d spectrophotometrically b y following the increase of the optical density at 262 m/~, as s h o w n in Fig. I. After measuring the rate of the auto-oxidation, the enzymic reaction was s t a r t e d b y adding a suitably diluted heart-muscle p r e p a r a t i o n to each cuvette. After the completion of the reaction, the s p e c t r u m was identical with t h a t of menadione, with b a n d s at 25 o, 262-263 and 34 ° m/z. The course of the oxidation shows first order kinetics, and the activity of the enzyme was expressed in t e r m s of the first order velocity constant, k'. Doubling the initial concentration of the K3H 2 did not affect k'. Since first order kinetics were obtained w i t h K3H ~, and zero order kinetics are found with comparable concentrations of D P N H 5, it is not possible to c o m p a r e the specific activities of the