YEASTALCOHOLDEHYDROGENASE REACTION
March 5 , 1957 [CONTRIBUTION NO.
745
FROM THE
DEPARTMENT O F CHEMISTRY,
1159
INDIANA UNIVERSITY]
Mechanisms of Enzyme-catalyzed Oxidation-Reduction Reactions. I. An Investigation of the Yeast Alcohol Dehydrogenase Reaction by Means of the Isotope Rate Effect1S2 BY
H. R. h l A H L E R
A N D JOYCE D O U G L A S
RECEIVED AUGUST8, 1956 The kinetics of the reversible dehydrogenation of ethanol by diphosphopyridine nucleotide, catalyzed by the alcohol dehydrogenase from yeast, have been examined in dilute phosphate buffer pH 7.6. Both protonated and deuterated reactants, viz. 1,l'-dideuteroethanol and DPND were used. The occurrence of appreciable isotope effects on the Michaelis constants for these two components ( b u t not for their reaction partners) and for the extrapolated maximal initial velocity in either direction has been demonstrated. T h e results of this and previous investigations are interpreted in terms of a mechanism which includes independent binding of substrate and coenzyme by Zn * .t and protein at the active site and a ratelimiting step ( a t high reactant concentration) with a transition state analogous to that postulated for the Meerwein-Pondorff-Oppenauer reaction.
The mechanism of action of certain pyridinenucleotide requiring dehydrogenases has been the subject of a number of recent papers, and attempts a t elucidation have made use of a variety of experimental and conceptional devices. These have taken the form of exhaustive kinetic studies employing both steady ~ t a t e ~and - ~ transient-stateg techniques, studies on the rate, nature and extent of binding of substrates, coenzymes and inbibitors,"-'0 non-enzymic model reactions11,12and a clear-cut demonstration that in the reaction catalyzed by these enzymes enzyme
SHz
+ Cot 7 S + CoH + H +
(l)I3
the transfer of hydrogen between substrate and the para position of the pyridine ring14 is direct and stereospecific. l5 In addition various possible over(1) Supported in p a r t by grant-in-aid BCH-9C from t h e American Cancer Society, on recommendation by t h e Committee on Growth, National Research Council, t o H . R . M . (2) Some of t h e experimental findings described in this report have been presented previously, a t t h e 129th Meeting of t h e American Chemical Society, Dallas, Texas, April, 1956, and a t the 47th Annual Meeting of t h e American Society of Biological Chemists, Atlantic City, N. J., April, 1956 ( F e d . Proc., 15, 307 (1956)). (3) H . Theorell a n d R. Bonnichsen, Acta Chem. Scand., 6, 1105 (1951); A. P. Nygaard and H . Theorell, i b i d . , 9, 1300, 1551 (1955). (4) H.Theorell, A. P. Nygaard a n d R . Bonnichsen, i b i d . , 9, 1148 (1955). ( 5 ) J. E . Hayes a n d S . F. Velick, J . Biol. Chem., 207, 225 (1'254). (6) M . T. Hakala, A. J. Glaid a n d G. W. Schwert, i b i d . , 221, 191 (1956). (7) J. B. Neilands, i b i d . , 199, 373 (1952); B. Chance a n d J. B. Neilands, i b i d . , 99, 383 (1952). ( 8 ) H. Theorell and B. Chance, A r l n Chcin. S c a i i d . , 6, 1127 (1951). (9) N . 0. Kaplan a n d M. M . Ciotti, J . Biol. C h e m . , 201, 785 (1953); N. 0. Kaplan. M . M . Ciotti and F. E. Stolzenbach, i b i d . , 211, 419 (1954); N. 0. Kaplan and M . M . Ciotti, i b i d . , 211, 431 (1954). (10) C. S. Vestling, H . Terayama, J. R . Florini and J . X . Baptist, F e d . Proc., 16, 375 (19.50). (11) A. J. Swallow, Biochem. J . , 61, I97 (1955); G . Stein and G. Stiassny, N a f u r c , 176, 734 (195.5). (12) R . H. Abeles a n d F. H . Westheimer, F e d . Pioc., 15, 208 (I95G). (13) T h e following abbreviations will be used: S, substrate of t h e general structure RIREC=O, here = CHaCHO; Co +-DPK, dipbosphopyridine nucleotide; CoH-DPNH, reduced diphosphopyridine nucleotide; S H D , CHsCDzOH; D P N D . paradeutero D P I i H ; e , molar extinction coefficient; E , enzyme; ki, rate constants; Ki, Michaelis and equilibrium constants; kn, K n , constants for reactants Containing deuterium; k H , K H , constants for reactants containing protium. (14) M . E. Pullman, A. San Pietro a n d S. P. Colowick, J . Biol. Chcm.. 206, 129 (1954). (15) For a review see B. Vennesland a n d F. H . Westheimet in "The Mechanism of Enzyme Action," ed. by W . D. McElroy and B. Glass, T h e Johns Hopkins Press, Baltimore, M d . , 1854.
all mechanisms for reaction 1 have been discussed and the applicable steady state rate equations der i ~ e d . ~As~ a, result ~ ~ of these investigations, considerable inroads have been made into the area of uncertainty concerning the mechanism of the overall reaction. On the other hand, relatively little is known concerning the mechanism of the hydrogen (or electron) transferring step proper and the transition state through which this transfer takes place. It occurred to us that the hydrogen isotope rate effect might be utilized to gain some information about the rate-limiting steps in enzyme-catalyzed oxidation-reduction reactions. For i t has been demonstrated experimentally and can be shown to hold from first principles a t least to a first approximation that, a t room temperature, a breaking of a C-H bond in the rate-limiting step will lead to a ratio k H / k D = 7 ; that a transition state in which the bonding to the hydrogen in the transition state is about as strong as that in the initial state will give a smaller ratio for k ~ / =k 1.4, ~ while so-called secondary isotope effects in reactions which do not involve the transfer of hydrogen a t all will be smaller still.l* Although the method has been employed successfully for the elucidation of the mechanism of organic oxidation-reduction reactions, l9 no application to isolated enzymatic reactions of this type has so far been reported. For our initial investigation we chose the alcohol dehydrogenase of yeast for the following reasons: (1) The enzyme can be obtained easily in crystalline form2o and is commercially available. ( 2 ) The kinetics of the enzyme-catalyzed reaction and the binding of the reaction partners by the cnzynie have been the subject of detailed investigationsRg6 which showed that (a) the kinetics are reasonably simple and straightforward, (b) the apparent dissociation constants for DPNH, D P N + and acetaldehyde are approximately equal to the experimentally determined Michaelis constants and ( e ) the enzyme can bind four molecules of coenzyme (16) H. L. Segal, J. F. Kachmar and P. D . Boyer, E n z y m o l . , 15, 187 (1952). (17) R . A . Alberty, THISJ O U R N A L , 75, 1928 (1953); Advairces i n E I I Z Y ~ R O17, ~ E 1Y (1956). , (18) A recent review is given b y K B. Wiberg, Chem. Reus., 66, 713 (1953). (19) F.11. Westheimer and N . Nicolaides, THISJ O U R N A L , 71, 25 ( I n i l l ) , I' 11. Westlieimer, Chem. Revs., 46, 419 (19.4'2). ('20) E Kacker, J . Bioi. C h e w . , 184, 313 (1950).
per molecule of proteiii a t equivalent sites; the binding sites for DPIV + are identical with those for DPNH. ( 3 ) Hydrogen transfer between substrate and coenzyme has been investigated by the use of deuterium-labeled reactants; i t was shown to be direct and stereospecific.16,21(4.) The enzyme has been shown to contain four gram-atoms of zinc per mole of protein. The metal has been tentatively iniplicated in enzymatic activity and coenzyiiie-binding.22 These latter results introduce an added parameter which must be taken into account when formulating ;t rcaction niechanisin. The enzyme catalyzes the reaction
+
R C H ~ O H DPS+ I JRCHO ( R = CHa in this investigation)
+ IIPSII + 11
+
](a)
13y nieans of kinetic investigations using 1,1'didcuteroethanol and DPND, it has been possible to confirni and extend some of the findings just described and to propose a structure for the traiisition state and a mechanism for the reaction consistent with all the experimental data so far available. These investigations form the basis for the present report.
Experimental Enzyme.-The enzyme used in these studies was a crystalline, comniercial product (\Vorthington Biochemical Corp.) recrystallized two additioiial times in this Laboratory.6 The stock suspension of the enzyme was kept a t - 1,5" arid diluted for kinetic experiments with a diluting medium t o yield tlie following final concentrations: enzyme protein, 10-40 p g. per ml.; crystalline bovine serum albumin (Armour or Pentes), 1 nig. per mi.; phosphate 0.001 li and cysteine 0.01 Jf, all adjustcd t o pH 7.6. Fnzyme made u p in this rnanncr is reasoriabl table a t t i o . New dilutions were made ever>-hour; rate r s under identical conditions showed the same initial rate of D P S H formatiun or disappearance a t the beginning and cnd of this period. Enzyme concentrations were deterrnined s~iectroph~~tonietrically at 280 nip using the molar extinction coefficient f e ) (1.89 x lo5 cTii.2 mole-') :ind molccuiar weight (150,000) reported by ITayes atid \.click.s Reactants.-Tlie D P N rras pu sctl f t - o i n tlie Sigtrr,t Cltetnical C o . I t \vas fourid to be 9 1)urefroti1 the optic,il density a t 260 n ~ p , using *~ e = 1 X 1 0 6 crn.2 Inole I . The purity of the sample used was 95YG as estimated fro,ii hydrosulfite reduction and 857{ as estimated from enzyrnat ic. reduction in the presence of excess alcohol, alcohol dehydrogenase and sernicarbazide at p H 7.6, or in the absence of sernicarbazide at pH 10.0. Similar results were also o ! tained with excess lactate and lactic dehydrogenase a t p i i 7.6. It seems likely t h a t this apparent discrepancy is rcfcrahlc t o the presence of approsimately 10Tc enzymatically inactive (x-imner of DPN.24 A11 measurements of D P X H formation or disappearance were made a t 340 mp, eZs = 6.22 X 1 0 6 c ~ i i .X~ mole-'. Solutions of acetaldehyde wcre prepared frcili and standardized prior to u s e b y reduction of aliquots in phosphate bufTer ,$H 7.0 in the presence o f escess DPXI-I and alcohol dch~,drogenase. Tlie D P S D used was prepared from the D P S sample indicated above by clieniical reduction bl- the method of Fisher, et 0 1 . 2 ~ Tlie reduction was performed in 100-nig. lots in 99.5% pure D,O (Stuart Oxygen C o . ) and could be shown t o be esscntially complete. The yield of isolated, -__ i s h e r , 13 IS. Gmn, I? T ' e n o e i l a n d a n d F. 11. XVe5tC h e m , 202, ns7 f l O i 3 1 illee a n d F.I*. I I o c h , TITISJ O L R N A L , 77, 8'11 ( 1 9 5 3 ) ; I'vor .Tu#:. A c u d .5ri , 4 1 , 327 (l%;,i!. (1'3) h. K o r n h r r g a n d I\E ' Pricer, J r . , Biorhenz.P r e b . , S , 20 (Iq:3), I'abst I , a ! X J r : l t O r i C S , ~ l l i r a v i o l e tn h w r p t i m Spectra of 5'-ribonu?leotides, h f i l w n i i k e e . IYiqc,, 1S.i.i ( 2 4 ) .2 0. Kt181:!n, R I . h1 C ' , n f t i I;. I< Stnl,rnl>nch a n d N. R. R , t r l i r i r , 'I't115 ~ ~ , i l ~ X A77, l . , 81; f Is>>), (2.;) 13. I, I I o r t c k e r a n d A . K o r n t ~ e r g ,J . Erol C l i e ! i i , 176, 38.5 ( I !)'18).
purified disodium DI'ND varied between 65 and 70(% or starting DPK on a weight basis. T h e dry weight purity of the reduced material varied between i 0 and 75Yc based o light absorption a t 340 m p . Of this absorption 9.5-10.5 was not destroyed b y enzymatic reoxidation of the reduc coenzyme by excess acetaldehyde and alcohol dehydrogenase a t p H 7.6, or b y pyruvate and lactic dehydrogenase or by cytochrome-c plus DPSII-cytochrome reductase. Since similar results were also obtained on analI1PI.j and of DPNH prepared from the discrepancy may he again referable t o t h a-isomer in the starting material. T h e D P S D prepared in this nianner was found not t o reduce either 2,R-diclilorophenolindophenol or cytochrome-c non-enzymatically a t ,a significant r a t e as determined b y measuring the cliange III light absorption a t 600 and 550 mp, respectively, and is thus relatively free of inorganic or organic reducing iinpuritics. In view of the reduction method employed in which hydrogen might be introduced only from the solid, anli>-drousreagents, the completeness of t h e reduction under these conditions and the demonstration t h a t the D P N I ) so formed contains one non-exchangable deuterium atom per inolecule,i5 the final product may be estiiiiated t o be contaminated b y
