Effect of pressure on deuterium isotope effects of formate dehydrogenase

High pressure causes biphasic effects on the oxidation of formate by yeast formate dehydrogenase as expressed on the kinetic parameter V/K, which measures substrate capture. Moderate pressure increases capture by accelerating hydride transfer. The transition state for hydride transfer has a smaller volume than the free formate plus the capturing form of enzyme, with DeltaV(double dagger) = -9.7 +/- 1.0 mL/mol. Pressures above 1.5 kbar decrease capture, reminiscent of effects on the conformational change associated with the binding of nicotinamide adenine dinucleotide (NAD(+)) to yeast alcohol dehydrogenase [Northrop, D. B., and Y. K. Cho (2000) Biochemistry 39, 2406-2412]. The collision complex, E-NAD(+), has a smaller volume than the more tightly bound reactant-state complex, E-NAD(+), with DeltaV = +83.4 +/- 5.2 mL/mol. A comparison of the effects of pressure on the oxidation of normal and deuteroformate shows that the entire isotope effect on hydride transfer, 2.73 +/- 0.20, arises solely from transition-state phenomena, as was also observed previously with yeast alcohol dehydrogense. In contrast, normal primary isotope effects arise solely from different zero-point energies in reactant states, and those that express hydrogen tunneling arise from a mixture of both reactant-state and transition-state phenomena. Moreover, pressure increases the primary intrinsic deuterium isotope effect, the opposite of what was observed with yeast alcohol dehydrogense. The lack of a decrease in the isotope effect is also contrary to empirical precedents from chemical reactions suspected of tunneling and to theoretical constructs of vibrationally enhanced tunneling in enzymatic reactions. Hence, this new experimental design penetrates transition states of enzymatic catalysis as never before, reveals the presence of phenomena foreign to chemical kinetics, and calls for explanations of how enzymes work beyond the tenants of physical organic chemistry.     

Role of internal thermodynamics in determining hydrogen tunneling in enzyme-catalyzed hydrogen transfer reactions.

Previous investigations have indicated a role for hydrogen tunneling in the yeast alcohol dehydrogenase catalyzed oxidation of benzyl alcohol [Cha, Y., Murray, C. J., & Klinman, J. P. (1989) Science 243, 1325] and the bovine plasma amine oxidase catalyzed oxidation of benzylamine [Grant, K.L., & Klinman, J. P. (1989) Biochemistry 28,6597]. In the present studies, values of protium to tritium and deuterium to tritium isotope effects and their temperature dependencies have been measured using ring-substituted substrates for yeast alcohol dehydrogenase and bovine plasma amine oxidase, revealing tunneling in each case. The results of these studies indicate that hydrogen tunneling is a general phenomenon and is not limited to enzyme reactions with degenerate energy levels for bound substrates and products. An analysis of internal thermodynamics in the yeast alcohol dehydrogenase reaction shows that tunneling occurs when delta H degrees is endothermic and that the degree of tunneling appears to increase as delta H degrees decreases toward zero.     

Effects of pressure on deuterium isotope effects of yeast alcohol dehydrogenase using alternative substrates

Hydrostatic pressure causes biphasic effects on the oxidation of alcohols by yeast alcohol dehydrogenase as expressed on the kinetic parameter V/K which measures substrate capture. Moderate pressure increases capture by activating hydride transfer, whose transition-state must therefore have a smaller volume than the free alcohol plus the capturing form of enzyme, with DeltaV(double dagger)=-30 mL mol(-1) for isopropanol. A comparison of these effects with those on the oxidation of deutero-isopropanol generates a monophasic decrease in the intrinsic isotope effect; therefore, the volume of activation for the transition-state of deuteride transfer must be even more negative, by 7.6 mL mol(-1). The pressure data extrapolate and factor the kinetic isotope effect into a semi-classical reactant-state component, with a null value of k(H)/k(D)=1, and a transition-state component of Q(H)/Q(D)=4, suggestive of hydrogen tunneling. Pressures above 1.5 kbar decrease capture by favoring a minor conformation of enzyme which binds nicotinamide adenine dinucleotide (NAD(+)) less tightly. This inactive conformation has a smaller volume than active E-NAD(+), with a difference of 74 mL mol(-1) and an equilibrium constant of 93 between them, at one atmosphere of pressure. These results are virtually identical to those obtained with benzyl alcohol and give credence to this method of analysis. Moreover, qualitatively similar results with greater pressure sensitivity but less precision are obtained using ethanol as a substrate, only with pressure driving the value of the isotope effect to a value less than (D)k=1.03 directly, without extrapolation. The ethanol data verify the most surprising finding of these studies, namely that the entire kinetic isotope effect arises from a transition-state phenomenon.     

Hydrogen tunneling in quinoproteins

It is now widely accepted that substrate C-H bond breakage by quinoprotein enzymes occurs by quantum mechanical tunneling. This paradigm shift in the conceptual framework for these reactions away from semi-classical transition state theory (i.e., including zero-point energy but with no tunneling correction) has been driven over recent years by experimental studies of the temperature dependence of kinetic isotope effects for these reactions in the TTQ-dependent enzymes methylamine dehydrogenase and aromatic amine dehydrogenase, which produced observations also inconsistent with the simple Bell correction model of tunneling. However, these data-specifically, the strong temperature dependence of reaction rates and the variable temperature dependence of kinetic isotope effects-are consistent with other tunneling models (denoted full tunneling models) in which protein and/or substrate fluctuations generate a configuration compatible with tunneling. These models accommodate substrate/protein (environment) fluctuations required to attain a configuration with degenerate quantum states and, when necessary, motion required to increase the probability of tunneling in these states. Furthermore, tunneling mechanisms in quinoproteins are supported by computational studies employing variational transition state theory with multidimensional tunneling corrections; these studies are also discussed in this review. Potential pitfalls in analyzing the temperature dependence of kinetic isotope effects as probes of tunneling are also discussed with reference to PQQ-dependent methanol dehydrogenase.     

Enzymatic H-transfer requires vibration-driven extreme tunneling

Enzymatic breakage of the substrate C-H bond by Methylophilus methyltrophus (sp. W3A1) methylamine dehydrogenase (MADH) has been studied by stopped-flow spectroscopy. The rate of reduction of the tryptophan tryptophylquinone (TTQ) cofactor has a large kinetic isotope effect (KIE = 16.8 +/- 0.5), and the KIE is independent of temperature. Analysis of the temperature dependence of C-H bond breakage revealed that extreme (ground state) quantum tunneling is responsible for the transfer of the hydrogen nucleus. Reaction rates are strongly dependent on temperature, indicating thermally induced, vibrational motion drives the H-transfer reaction. The data provide direct experimental evidence for enzymatic bond breakage by extreme tunneling driven by vibrational motion of the protein scaffold. The results demonstrate that classical transition state theory and its tunneling derivatives do not adequately describe this enzymatic reaction.     

Environmentally coupled hydrogen tunneling. Linking catalysis to dynamics.

Many biological C-H activation reactions exhibit nonclassical kinetic isotope effects (KIEs). These nonclassical KIEs are too large (kH/kD > 7) and/or exhibit unusual temperature dependence such that the Arrhenius prefactor KIEs (AH/AD) fall outside of the semiclassical range near unity. The focus of this minireview is to discuss such KIEs within the context of the environmentally coupled hydrogen tunneling model. Full tunneling models of hydrogen transfer assume that protein or solvent fluctuations generate a reactive configuration along the classical, heavy-atom coordinate, from which the hydrogen transfers via nuclear tunneling. Environmentally coupled tunneling also invokes an environmental vibration (gating) that modulates the tunneling barrier, leading to a temperature-dependent KIE. These properties directly link enzyme fluctuations to the reaction coordinate for hydrogen transfer, making a quantum view of hydrogen transfer necessarily a dynamic view of catalysis. The environmentally coupled hydrogen tunneling model leads to a range of magnitudes of KIEs, which reflect the tunneling barrier, and a range of AH/AD values, which reflect the extent to which gating modulates hydrogen transfer. Gating is the primary determinant of the temperature dependence of the KIE within this model, providing insight into the importance of this motion in modulating the reaction coordinate. The potential use of variable temperature KIEs as a direct probe of coupling between environmental dynamics and the reaction coordinate is described. The evolution from application of a tunneling correction to a full tunneling model in enzymatic H transfer reactions is discussed in the context of a thermophilic alcohol dehydrogenase and soybean lipoxygenase-1.     

Transition-state structures for N-glycoside hydrolysis of AMP by acid and by AMP nucleosidase in the presence and absence of allosteric activator

The mechanism of acid and enzymatic hydrolysis of the N-glycosidic bond of AMP has been investigated by fitting experimentally observed kinetic isotope effects [Parkin, D. W., & Schramm, V. L. (1987) Biochemistry (preceding paper in this issue)] to calculated kinetic isotope effects for proposed transition-state structures. The sensitivity of the transition-state calculations was tested by "arying the transition-state structure and comparing changes in the calculated kinetic isotope effects with the experimental values of the isotope effect measurements. The kinetic isotope effects for the acid-catalyzed hydrolysis of AMP are best explained by a transition state with considerable oxycarbonium character in the ribose ring, significant bonding remaining to the departing adenine ring, participation of a water nucleophile, and protonation of the adenine ring. A transition-state structure without preassociation of the water nucleophile cannot be eliminated by the data. Enzymatic hydrolysis of the N-glycosidic bond of AMP by AMP nucleosidase from Azotobacter vinelandii was analyzed in the absence and presence of MgATP, the allosteric activator that increases Vmax approximately 200-fold. The transition states for enzyme-catalyzed hydrolysis that best explain the kinetic isotope effects involve early SN1 transition states with significant bond order in the glycosidic bond and protonation of the adenine base. The enzyme enforces participation of an enzyme-bound water molecule, which has weak bonding to C1' in the transition state. Activation of AMP nucleosidase by MgATP causes the bond order of the glycosidic bond in the transition state to increase significantly. Hyperconjugation in the ribosyl group is altered by enzymatic stabilization of the oxycarbonium ion. This change is consistent with the interaction of an amino acid on the enzyme. Together, these changes stabilize a carboxonium-like transition-state complex that occurs earlier in the reaction pathway than in the absence of allosteric activator. In addition to the allosteric changes that alter transition-state structure, the presence of other inductive effects that are unobserved by kinetic isotope measurements is also likely to increase the catalytic rate.     

Probes of hydrogen tunneling with horse liver alcohol dehydrogenase at subzero temperatures

The temperature dependence of steady-state kinetics has been studied with horse liver alcohol dehydrogenase (HLADH) using protonated and deuterated benzyl alcohol as substrates in methanol/water mixtures between +3 and -50 degrees C. Additionally, the competitive isotope effects, k(H)/k(T) and k(D)/k(T), were measured. The studies indicate increasing kinetic complexity for wild-type HLADH at subzero temperatures. Consistent with earlier findings at 25 degrees C [Bahnson et al. (1993) Biochemistry 31, 5503], the F93W mutant shows much less kinetic complexity than the wild-type enzyme between 3 and -35 degrees C. An analysis of noncompetitive deuterium isotope effects and competitive tritium isotope effects leads to the conclusion that the reaction of F93W involves substantial hydrogen tunneling down to -35 degrees C. The effect of methanol on kinetic properties for the F93W mutant was analyzed, showing a dependence of competitive KIEs on the NAD(+) concentration. This indicates a more random bi--bi kinetic mechanism, in comparison to an ordered bi-bi kinetic mechanism in water. Although MeOH also affects the magnitude of the reaction rates and, to some extent, the observed KIEs, the ratio of ln k(H)/k(T) to ln k(D)/k(T) for primary isotope effects has not changed in methanol, and we conclude little or no change in kinetic complexity. Importantly, the degree of tunneling, as shown from the relationship between the secondary k(H)/k(T) and k(D)/k(T) values, is the same in water and MeOH/water mixtures, implicating similar trajectories for H transfer in both solvents. In a recent study of a thermophilic alcohol dehydrogenase [Kohen et al. (1999) Nature 399, 496], it was shown that decreases in temperatures below a transition temperature lead to decreased tunneling. This arises because of a change in protein dynamics below a break point in enzyme activity [Kohen et al. (2000) J. Am. Chem. Soc. 122, 10738-10739]. For the mesophilic HLADH described herein, an opposite trend is observed in which tunneling increases at subzero temperatures. These differences are attributed to inherent differences in tunneling probabilities between 0 and 100 degrees C vs subzero temperatures, as opposed to fundamental differences in protein structure for enzymes from mesophilic vs thermophilic sources. We propose that future investigations of the relationship between protein flexibility and hydrogen tunneling are best approached using enzymes from thermophilic sources.     

Probes of a role for remote binding interactions on hydrogen tunneling in the horse liver alcohol dehydrogenase reaction

A tunneling contribution to hydride transfer has been demonstrated previously in the oxidation of benzyl alcohol catalyzed by an active-site mutant (F93W) of horse liver alcohol dehydrogenase (LADH) [Bahnson, B. J., et al. (1993) Biochemistry 32, 5503-5507]. Mutation of a residue that lies directly behind the nicotinamide ring of the bound cofactor has further shown that side-chain bulk can contribute to catalytic efficiency and tunneling in a correlated fashion [Bahnson, B. J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 12797-12802]. Second site mutations of F93W have now been made at positions more remote from the active site. In particular, we have focused on an isoleucine residue that interacts with the adenine moiety of the NAD(+) cofactor, 20 A from the nicotinamide ring. Replacement of this remote residue with glycine (F93W:I224G), alanine (F93W:I224A), valine (F93W:I224V), and leucine (F93W:I224L) is concluded to destabilize the binding of NAD(+). All double mutants exhibited a K(M) for NAD(+) that is 2-25 times higher than that for the F93W enzyme. However, neither the catalytic efficiency for turnover of benzyl alcohol [k(cat)/K(M(benzyl alcohol))] nor the relationship between the secondary k(H)/k(T) and k(D)/k(T) isotope effects for benzyl alcohol oxidation was significantly affected. The lack of differences observed in the isotope effects indicates that these mutations have little effect on the extent of hydrogen tunneling in the reaction. The complete removal of the side chain at position 224 in the F93W:I224G enzyme resulted in a less than 5% decrease in the ratio of the secondary isotope effects, maintaining the ratio above the semiclassical limit for the indication of tunneling in the reaction. By contrast, K(i) for NAD(+) increased 60-fold for this mutant. The results obtained with F93W:I224G are consistent with remote interactions that affect the association and binding of cofactor in a reactive conformation. However, once this conformation is achieved, hydride transfer and its tunneling component proceed as with the single F93W mutant enzyme, uninfluenced by the remote mutation. Replacement of other side chains, with alpha-carbon positions from about 8 to over 20 A from the C4 position of the nicotinamide ring, demonstrated a similar insensitivity of k(cat)/K(M(benzyl alcohol)) to protein modification. Comparison to earlier studies with active-site mutants of LADH implicates a role for proximal, but not distal, side chains in the modulation of hydrogen tunneling for this enzyme.     

Nature of hydrogen transfer in soybean lipoxygenase 1: separation of primary and secondary isotope effects

Previous measurements of the kinetics of oxidation of linoleic acid by soybean lipoxygenase 1 have indicated very large deuterium isotope effects, but have not been able to distinguish the primary isotope effect from the alpha-secondary effect. To address this question, singly deuterated linoleic acid was prepared, and enantiomerically resolved using the enzyme itself. Noncompetitive measurements of the primary deuterium isotope effect give a value of ca. 40 which is temperature-independent. The enthalpy of activation is low and isotope-independent, and there is a large isotope effect on the Arrhenius prefactor. A very large apparent secondary isotope effect (ca. 2.1) is measured with deuterium in the primary position, but a greatly reduced value (1.1) is observed with protium in the primary position. Mutagenesis of the active site leads to a significant reduction in k(cat) and perturbed isotope effects, in particular, a secondary effect of 5.6 when deuterium is in the primary position. The anomalous secondary isotope effects are shown to arise from imperfect stereoselectivity of hydrogen abstraction which, for the mutant, is attributed to a combination of inverse substrate binding and increased flexibility at the reactive carbon. After correction, a very large primary (76-84) and small secondary (1.1-1.2) kinetic isotope effects are calculated for both mutant and wild-type enzymes. The weight of the evidence is taken to favor hydrogen tunneling as the primary mechanism of hydrogen transfer.     

Rational design of transition-state analogues as potent enzyme inhibitors with therapeutic applications.

The structures of the transition states for a variety of enzyme-catalyzed ribosyl group transfer reactions, determined by computational evaluation of multiple tritium and heavy atom kinetic isotope effects on these enzymatic reactions, have been found to show a considerable variation in the extent of bond cleavage at the ribosyl anomeric carbon. The calculated transition-state structures have been used to guide the design of high-affinity transition-state analogue inhibitors for 5'-methylthioadenosine nucleosidases with potential as therapeutic agents.     

Effects of high pressure on enzymatic activity

Effects of high pressure on enzymatic reactions are poised to revolutionize enzyme kinetics. The reason for this is that experimental designs are at hand to separate effects on equilibria between reactant states from effects on catalytic transition states and both yield new information. The first of the former runs contrary to Pauling's hypothesis that substrates are bound more tightly in the transition state, while the latter penetrates the 'black box' of catalysis, the stabilized transition state itself, and returns a precise measure of a physical parameter, deltaV. This in turn opens the door to new forms of structure-activity relationships. The first of these has been described, the effect of pressure on isotope effects, with the surprising finding that the entire isotope effect comes from a transition state phenomenon such as quantum mechanical hydrogen tunneling.     

De novo design and utilization of photolabile caged substrates as probes of hydrogen tunneling with horse liver alcohol dehydrogenase at sub-zero temperatures: a cautionary note

In order to understand the influence of protein dynamics on enzyme catalysis and hydrogen tunneling, the horse liver alcohol dehydrogenase (HLADH) catalyzed oxidation of benzyl alcohol was studied at sub-zero temperatures. Previous work showed that wild type HLADH has significant kinetic complexity down to -50 degrees C due to slow binding and loss of substrate [S.-C. Tsai, J.P. Klinman, Biochemistry, 40 (2001) 2303]. A strategy was therefore undertaken to reduce kinetic complexity at sub-zero temperatures, using a photolabile (caged) benzyl alcohol that prebinds to the enzyme and yields the active substrate upon photolysis. By computer modeling, a series of caged alcohols were designed de novo, synthesized, and characterized with regard to photolysis and binding properties. The o-nitrobenzyl ether 15, with a unique long tail, was found to be most ideal. At sub-zero temperatures in 50% MeOH, a two-phase kinetic trace and a rate enhancement by the use of 15 were observed. Despite the elimination of substrate binding as a rate-limiting step, the use of caged benzyl alcohol does not produce a measurable H/D kinetic isotope effect. Unexpectedly, the observed fast phase corresponds to multiple enzyme turnovers, based on the stoichiometry of the substrate to enzyme. Possible side reactions and their effects, such as the re-oxidation of bound NADH and the dissipation of photo-excitation energy, may offer an explanation for the observed multiple-turnovers. The lack of observable deuterium isotope effects offers a cautionary note for the application of caged substrates to isolate and study chemical steps of enzyme reactions, particularly when NADH is involved in the reaction pathway.     

Theoretical evaluation of the hydrogen kinetic isotope effect on the first step of the methylmalonyl-CoA mutase reaction

We have calculated hydrogen kinetic isotope effects (KIEs) for the first step of the methylmalonyl-CoA mutase reaction, including multidimensional tunneling correction at the zero curvature (ZCT) level, and compared them with the experimental values. Both alternative mechanisms of this step, concerted and stepwise, can be accommodated. It turned out to be essential to include Arg207 hydrogen-bonded to the reactant in the mechanism predicting simultaneous breaking of the Co-C bond of AdoCbl and hydrogen atom transfer. The consequence of the stepwise mechanism is a much larger facilitation of the homolytic dissociation of the carbon-cobalt bond by the enzyme than currently appreciated; our results suggest lowering of the activation energy by about 23 kcal mol(-1). We have also shown that large hydrogen KIEs of tunneling origin do not necessarily break the Swain-Schaad equation. Furthermore, when this equation does not hold, the exponent may be smaller in the presence of tunneling than it is at the semi-classical limit, indicating that nonclassical behavior may be a more common phenomenon than expected.     

The mechanism of the proton transfer: an outline

A brief summary of the principal notions of the quantum-mechanical theory of the charge transfer reactions has been presented. In the framework of this theory, the mechanism of the proton transfer consists in the classical medium reorganization that equalizes the proton energy levels in the initial and final states, and a consequent proton transfer via a quantum-mechanical underbarrier transition. On the basis of this mechanism, factors influencing the proton transfer probability, and hence kinetic isotope effect, have been discussed; among them are the optimum tunneling distance, the involvement of the excited vibrational states, etc. Semi-classical and quantum-mechanical treatments of the Swain-Schaad relations have been compared. Some applications to enzymatic proton-transfer reactions have been described.     

Secondary 15N isotope effects on the reactions catalyzed by alcohol and formate dehydrogenases

Secondary 15N isotope effects at the N-1 position of 3-acetylpyridine adenine dinucleotide have been determined, by using the internal competition technique, for horse liver alcohol dehydrogenase (LADH) with cyclohexanol as a substrate and yeast formate dehydrogenase (FDH) with formate as a substrate. On the basis of less precise previous measurements of these 15N isotope effects, the nicotinamide ring of NAD has been suggested to adopt a boat conformation with carbonium ion character at C-4 during hydride transfer [Cook, P. F., Oppenheimer, N. J. & Cleland, W. W. (1981) Biochemistry 20, 1817]. If this mechanism were valid, as N-1 becomes pyramidal an 15N isotope effect of up to 2-3% would be observed. In the present study the equilibrium 15N isotope effect for the reaction catalyzed by LADH was measured as 1.0042 +/- 0.0007. The kinetic 15N isotope effect for LADH catalysis was 0.9989 +/- 0.0006 for cyclohexanol oxidation and 0.997 +/- 0.002 for cyclohexanone reduction. The kinetic 15N isotope effect for FDH catalysis was 1.004 +/- 0.001. These values suggest that a significant 15N kinetic isotope effect is not associated with hydride transfer for LADH and FDH. Thus, in contrast with the deformation mechanism previously postulated, the pyridine ring of the nucleotide apparently remains planar during these dehydrogenase reactions.     

Quantum mechanical hydrogen tunneling in bacterial copper amine oxidase reaction

A key step decisively affecting the catalytic efficiency of copper amine oxidase is stereospecific abstraction of substrate alpha-proton by a conserved Asp residue. We analyzed this step by pre-steady-state kinetics using a bacterial enzyme and stereospecifically deuterium-labeled substrates, 2-phenylethylamine and tyramine. A small and temperature-dependent kinetic isotope effect (KIE) was observed with 2-phenylethylamine, whereas a large and temperature-independent KIE was observed with tyramine in the alpha-proton abstraction step, showing that this step is driven by quantum mechanical hydrogen tunneling rather than the classical transition-state mechanism. Furthermore, an Arrhenius-type preexponential factor ratio approaching a transition-state value was obtained in the reaction of a mutant enzyme lacking the critical Asp. These results provide strong evidence for enzyme-enhanced hydrogen tunneling. X-ray crystallographic structures of the reaction intermediates revealed a small difference in the binding mode of distal parts of substrates, which would modulate hydrogen tunneling proceeding through either active or passive dynamics.     

Vibrationally enhanced hydrogen tunneling in the Escherichia coli thymidylate synthase catalyzed reaction

The enzyme thymidylate synthase (TS) catalyzes a complex reaction that involves forming and breaking at least six covalent bonds. The physical nature of the hydride transfer step in this complex reaction cascade has been studied by means of isotope effects and their temperature dependence. Competitive kinetic isotope effects (KIEs) on the second-order rate constant (V/K) were measured over a temperature range of 5-45 degrees C. The observed H/T ((T)V/K(H)) and D/T ((T)V/K(D)) KIEs were used to calculate the intrinsic KIEs throughout the temperature range. The Swain-Schaad relationships between the H/T and D/T V/K KIEs revealed that the hydride transfer step is the rate-determining step at the physiological temperature of Escherichia coli (20-30 degrees C) but is only partly rate-determining at elevated and reduced temperatures. H/D KIE on the first-order rate constant k(cat) ((D)k = 3.72) has been previously reported [Spencer et al. (1997) Biochemistry 36, 4212-4222]. Additionally, the Swain-Schaad relationships between that (D)k and the V/K KIEs reported here suggested that at 20 degrees C the hydride transfer step is the rate-determining step for both rate constants. Intrinsic KIEs were calculated here and were found to be virtually temperature independent (DeltaE(a) = 0 within experimental error). The isotope effects on the preexponential Arrhenius factors for the intrinsic KIEs were A(H)/A(T) = 6.8 +/- 2.8 and A(D)/A(T) = 1.9 +/- 0.25. Both effects are significantly above the semiclassical (no-tunneling) predicted values and indicate a contribution of quantum mechanical tunneling to this hydride transfer reaction. Tunneling correction to transition state theory would predict that these isotope effects on activation parameters result from no energy of activation for all isotopes. Yet, initial velocity measurements over the same temperature range indicate cofactor inhibition and result in significant activation energy on k(cat) (4.0 +/- 0.1 kcal/mol). Taken together, the temperature-independent KIEs, the large isotope effects on the preexponential Arrhenius factors, and a significant energy of activation all suggest vibrationally enhanced hydride tunneling in the TS-catalyzed reaction.     

Unusual origins of isotope effects in enzyme-catalysed reactions

High hydrostatic pressure is a neglected tool for probing the origins of isotope effects. In chemical reactions, normal primary deuterium isotope effects (DIEs) arising solely from differences in zero point energies are unaffected by pressure; but some anomalous isotope effects in which hydrogen tunnelling is suspected are partially suppressed. In some enzymatic reactions, high pressure completely suppresses the DIE. We have now measured the effects of high pressure on the parallel 13C heavy atom isotope effect of yeast alcohol dehydrogenase and found that it is also suppressed by high pressure and, similarly, suppressed in its entirety. Moreover, the volume changes associated with the suppression of both deuterium and heavy atom isotope effects are virtually identical. The equivalent decrease in activation volumes for hydride transfer, when one mass unit is added to the carbon end of a scissile C-H bond as when one mass unit is added to the hydrogen end, suggests a common origin. Given that carbon is highly unlikely to undergo tunnelling, it follows that hydrogen is not doing so either. The origin of these isotope effects must lie elsewhere. We offer protein domain motions as a possibility.