Kinetic isotope effects on aromatic and benzylic hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase as probes of chemical mechanism and reactivity

Phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine to tyrosine. In this study, we used deuterium kinetic isotope effects to probe the chemical mechanisms of aromatic and benzylic hydroxylation to compare the reactivities of bacterial and eukaryotic aromatic amino acid hydroxylases. The (D) k cat value for the reaction of CvPheH with [(2)H 5]phenylalanine is 1.2 with 6-methyltetrahydropterin and 1.4 with 6,7-dimethyltetrahydropterin. With the mutant enzyme I234D, the (D) k cat value decreases to 0.9 with the latter pterin; this is likely to be the intrinsic effect for addition of oxygen to the amino acid. The isotope effect on the subsequent tautomerization of a dienone intermediate was determined to be 5.1 by measuring the retention of deuterium in tyrosine produced from partially deuterated phenylalanine; this large isotope effect is responsible for the normal effect on k cat. The isotope effect for hydroxylation of the methyl group of 4-CH 3-phenylalanine, obtained from the partitioning of benzylic and aromatic hydroxylation products, is 10. The temperature dependence of this isotope effect establishes the contribution of hydrogen tunneling to benzylic hydroxylation by this enzyme. The results presented here provide evidence that the reactivities of the prokaryotic and eukaryotic hydroxylases are similar and further define the reactivity of the iron center for the family of aromatic amino acid hydroxylases.     

Evidence for coupled motion and hydrogen tunneling of the reaction catalyzed by glutamate mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through organic radical intermediates generated by homolytic fission of the coenzyme's unique cobalt-carbon bond. These enzymes are part of a larger family of enzymes that catalyze radical chemistry in which a key step is the abstraction of a hydrogen atom from an otherwise inert substrate. To gain insight into the mechanism of hydrogen transfer, we previously used pre-steady-state, rapid-quench techniques to measure the alpha-secondary tritium kinetic and equilibrium isotope effects associated with the formation of 5'-deoxyadenosine when glutamate mutase was reacted with [5'-(3)H]adenosylcobalamin and L-glutamate. We showed that both the kinetic and equilibrium isotope effects are large and inverse, 0.76 and 0.72, respectively. We have now repeated these measurements using glutamate deuterated in the position of hydrogen abstraction. The effect of introducing a primary deuterium kinetic isotope effect on the hydrogen transfer step is to reduce the magnitude of the secondary kinetic isotope effect to a value close to unity, 1.05 +/- 0.08, whereas the equilibrium isotope effect is unchanged. The significant reduction in the secondary kinetic isotope effect is consistent with motions of the 5'-hydrogen atoms being coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling.     

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.     

Insights into the mechanism of flavoprotein-catalyzed amine oxidation from nitrogen isotope effects on the reaction of N-methyltryptophan oxidase

The mechanism of N-methyltryptophan oxidase, a flavin-dependent amine oxidase from Escherichia coli, was studied using a combination of kinetic isotope effects and theoretical calculations. The 15(kcat/Km) kinetic isotope effect for sarcosine oxidation is pH-dependent with a limiting value of 0.994-0.995 at high pH. Density functional theory calculations on model systems were used to interpret these isotope effects. The isotope effects are inconsistent with proposed mechanisms involving covalent amine-flavin adducts but cannot by themselves conclusively distinguish between some discrete electron-transfer mechanisms and a direct hydride-transfer mechanism, although the latter mechanism is more consistent with the energetics of the reaction.     

pH and kinetic isotope effects on sarcosine oxidation by N-methyltryptophan oxidase

N-Methyltryptophan oxidase (MTOX), a flavoenzyme from Escherichia coli, catalyzes the oxidative demethylation of secondary amino acids such as N-methyltryptophan or N-methylglycine (sarcosine). MTOX is one of several flavin-dependent amine oxidases whose chemical mechanism is still debated. The kinetic properties of MTOX with the slow substrate sarcosine were determined. Initial rate data are well-described by the equation for a ping-pong kinetic mechanism, in that the V/K(O)()2 value is independent of the sarcosine concentration at all accessible concentrations of oxygen. The k(cat)/K(sarc) pH profile is bell-shaped, with pK(a) values of 8.8 and about 10; the latter value matches the pK(a) value of the substrate nitrogen. The k(cat) pH profile exhibits a single pK(a) value of 9.1 for a group that must be unprotonated for catalysis. There is no significant solvent isotope effect on the k(cat)/K(sarc) value. With N-methyl-(2)H(3)-glycine as the substrate, there is a pH-independent kinetic isotope effect on k(cat), k(cat)/K(sarc), and the rate constant for flavin reduction, with an average value of 7.2. Stopped-flow spectroscopy with both the protiated and deuterated substrate failed to detect any intermediates between the enzyme-substrate complex and the fully reduced enzyme. These results are used to evaluate proposed chemical mechanisms.     

Detailed dissection of a new mechanism for glycoside cleavage: alpha-1,4-glucan lyase

The unusual enzyme, Gracilariopsis alpha-1,4-glucan lyase of the sequence-related glycoside hydrolase family 31, cleaves the glycosidic bond of alpha-1,4-glucans via a beta-elimination reaction involving a covalent glycosyl-enzyme intermediate (Lee, S. S., Yu, S., and Withers, S. G. (2002) J. Am. Chem. Soc. 124, 4948-4949). The classical bell-shaped pH dependence of k(cat)/K(m) indicates two ionizable groups in the active site with apparent pK(a) values of 3.05 and 6.66. Br?nsted relationships of log k(cat) versus pK(a) and log(k(cat)/K(m)) versus pK(a) for a series of aryl glucosides both show a linear monotonic dependence on leaving group pK(a) with low beta(lg) values of 0.32 and 0.33, respectively. The combination of these low beta(lg) values with large secondary deuterium kinetic isotope effects (k(H)/k(D) = 1.16 - 1.19) on the first step indicate a glycosylation step with substantial glycosidic bond cleavage and proton donation to the leaving group oxygen at the transition state. Developed oxocarbenium ion character of the transition state is also suggested by the potent inhibition afforded by acarbose and 1-deoxynojirimycin (K(i) = 20 and 130 nM, respectively) and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-alpha-D-glucopyranosyl fluoride, was the second elimination step shown to be rate-limiting. The large alpha-secondary deuterium kinetic isotope effect (k(H)/k(D) = 1.23) at C-1 and the small primary deuterium kinetic isotope effect (k(H)/k(D) = 1.92) at C-2 confirm an E2 mechanism with strong E1 character for this second step. This considerable structural and mechanistic similarity with retaining alpha-glucosidases is clear evidence for the evolution of an enzyme mechanism within the family.     

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

Use of nitrogen-15 and deuterium isotope effects to determine the chemical mechanism of phenylalanine ammonia-lyase

Phenylalanine ammonia-lyase has been shown to catalyze the elimination of ammonia from the slow alternate substrate 3-(1,4-cyclohexadienyl)alanine by an E1 cb mechanism with a carbanion intermediate. This conclusion resulted from comparison of 15N isotope effects with deuterated (0.9921) and unlabeled substrates (1.0047), and a deuterium isotope effect of 2.0 from dideuteration at C-3, with the equations for concerted, carbanion, and carbonium ion mechanisms. The 15N equilibrium isotope effect on the addition of the substrate to the dehydroalanine prosthetic group on the enzyme is 0.979, while the kinetic 15N isotope effect on the reverse of this step is 1.03-1.04 and the intrinsic deuterium isotope effect on proton removal is in the range 4-6. Isotope effects with phenylalanine itself are small (15N ones of 1.0021 and 1.0010 when unlabeled or 3-dideuterated and a deuterium isotope effect of 1.15) but are consistent with the same mechanism with drastically increased commitments, including a sizable external one (i.e., phenylalanine is sticky). pH profiles show that the amino group of the substrate must be unprotonated to react but that a group on the enzyme with a pK of 9 must be protonated, possibly to catalyze addition of the substrate to dehydroalanine. Incorrectly protonated enzyme-substrate complexes do not form. Equilibrium 15N isotope effects are 1.016 for the deprotonation of phenylalanine or its cyclohexadienyl analogue, 1.0192 for deprotonation of NH4+, 1.0163 for the conversion of the monoanion of phenylalanine to NH3, and 1.0138 for the conversion of the monoanion of aspartate to NH4+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of ionizable residues in the catalytic mechanism of tryptophan synthase from Salmonella typhimurium

The tryptophan synthase alpha2beta2 complex catalyzes the last two steps in the biosynthesis of l-tryptophan in bacteria, plants, and fungi, the conversion of indole-3-glycerol phosphate and l-serine to l-tryptophan, glyceraldehyde 3-phosphate, and water. The beta-subunit binds pyridoxal 5'-phosphate and catalyzes the beta-replacement reaction with serine and indole. Structural, spectral, and kinetic studies indicate that different monovalent cations stabilize the alternative enzyme conformations and equilibrium distribution of the internal, external, and alpha-aminoacrylate Schiff base. To improve our understanding of the role of monovalent cations, the pH dependence of steady-state and pre-steady-state kinetic parameters and primary kinetic deuterium isotope effects were measured in the presence of l-serine and [alpha-2H]-l-serine in the absence and presence of Na+, K+, and Cs+. For the interpretation of the data obtained in this study, it was necessary to re-interpret a number of results published previously. Overall, data suggest that the enzyme exists in two conformers that equilibrate slowly either in the absence of substrates and monovalent cations or in the presence of K+ or Cs+, whereas they equilibrate faster in the presence of Na+. The rate of interconversion of the conformers increases as a group on the enzyme with a pKa of approximately 8 becomes deprotonated. The pH dependence of deuterium isotope effects is suggestive of a mechanism in which a pH-dependent conformational change that closes the active site precedes the chemical steps, likely a result of formation of one or more salt bridges. As the pH increases, the reaction becomes more committed to proceed to products, which causes the deuterium isotope effect to decrease to a value of unity at high pH. The closure of the site is modulated by the different monovalent cations and is fastest in the presence of Na+, which exhibits the maximum isotope effect of 5.7 (likely the intrinsic effect) on V/Kserine, and slowest in the presence of Cs+, which exhibits the smallest isotope effect of approximately 1.5. The isotope effect on V, in all cases, indicates a contribution to rate limitation from steps in the second half of the reaction. Finally, in the presence of Na+, the steady-state isotope effect on V is greater than that on the pre-steady-state rate constant for decay of the external Schiff base, suggesting that the rate of conversion of the two conformers of the internal aldimine contributes to the pre-steady-state rate, but not the steady-state rate because the high serine concentration traps the enzyme in the active E-serine complex before it can decay to the less active form.     

Kinetic and chemical mechanisms of the fabG-encoded Streptococcus pneumoniae beta-ketoacyl-ACP reductase

Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.     

Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme

The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dependent oxidative decarboxylation of L-malate to give pyruvate and CO2, with NAD+ as the oxidant. Alpha-secondary tritium kinetic isotope effects were measured with NAD+ or APAD+ and L-malate-2-H(D) and several different divalent metal ions. The alpha-secondary tritium kinetic isotope effects are slightly higher than 1 with NAD+ and L-malate as substrates, much larger than the expected inverse isotope effect for a hybridization change from sp2 to sp3. The alpha-secondary tritium kinetic isotope effects are reduced to values near 1 with L-malate-2-D as the substrate, regardless of the metal ion that is used. Data suggest the presence of quantum mechanical tunneling and coupled motion in the malic enzyme reaction when NAD+ and malate are used as substrates. Isotope effects were also measured using the D/T method with NAD+ and Mn2+ as the substrate pair. A Swain-Schaad exponent of 2.2 (less than the value of 3.26 expected for strictly semiclassical behavior) is estimated, suggesting the presence of other slow steps along the reaction pathway. With APAD+ and Mn2+ as the substrate pair, inverse alpha-secondary tritium kinetic isotope effects are observed, and a Swain-Schaad exponent of 3.3 is estimated, consistent with rate-limiting hydride transfer and no quantum mechanical tunneling or coupled motion. Data are discussed in terms of the malic enzyme mechanism and the theory developed by Huskey for D/T isotope effects as an indicator of tunneling [Huskey, W. P. (1991) J. Phys. Org. Chem. 4, 361-366].     

The use of isotope effects to determine enzyme mechanisms

Isotope effects are one of the most powerful kinetic tools for determining enzyme mechanisms. There are three methods of measurement. First, one can compare reciprocal plots with labeled and unlabeled substrates. The ratio of the slopes is the isotope effect on V/K, and the ratio of the vertical intercepts is the isotope effect on V(max). This is the only way to determine V(max) isotope effects, but is limited to isotope effects of 5% or greater. The second method is internal competition, where the labeled and unlabeled substrates are present at the same time and the change in their ratio in residual substrate or in product is used to calculate an isotope effect, which is that on V/K of the labeled reactant. This is the method used for tritium or (14)C, or with the natural abundances of (13)C, (15)N, or (18)O. The third method involves perturbations from equilibrium when a labeled substrate and corresponding unlabeled product are present at chemical equilibrium. This also gives just an isotope effect on V/K for the labeled reactant. The chemistry is typically not fully rate limiting, so that the isotope effect on V/K is given by: (x)(V/K)=((x)k+c(f)+c(r)(x)K(eq))/(1+c(f)+c(r)) where x defines the isotope (D, T, 13, 15, 18 for deuterium, tritium, (13)C, (15)N, or (18)O), and (x)(V/K), (x)k, and (x)K(eq) are the observed isotope effect, the intrinsic one on the chemical step, and the isotope effect on the equilibrium constant, respectively. The constants c(f) and c(r) are commitments in forward and reverse directions, and are the ratio of the rate constant for the chemical reaction and the net rate constant for release from the enzyme of the varied substrate (direct comparison) or labeled substrate (internal competition and equilibrium perturbation) for c(f), or the first product released or the one involved in the perturbation for c(r). The intrinsic isotope effect, (x)k, can be estimated by comparing deuterium and tritium isotope effects on V/K, or by comparing the deuterium isotope effect with (13)C ones with deuterated and undeuterated substrates. Adding a secondary deuterium isotope effect and its effect on the (13)C one can give an exact solution for all intrinsic isotope effects and commitments. The effect of deuteration on a (13)C isotope effect allows one to tell if the two isotope effects are on the same or different steps. Applications of these methods to several enzyme systems will be presented.     

Rapid-scan stopped-flow studies of the pH dependence of the reaction between mercuric reductase and NADPH

The reaction of NADPH with the flavoenzyme mercuric reductase has been studied by rapid-scan stopped-flow spectrophotometry at 5 degrees C in the pH range 5.1-9.5. An intermediate formed within the dead time of the apparatus, and proposed to be an NADPH complex of oxidized enzyme, has an almost pH-independent spectrum. At pH 5.1 the formation of this species is followed by a rapid bleaching (k = 145 s-1) of the main flavin absorption band at 455 nm concomitantly with an absorbance increase around 395 nm. This process, which has a kinetic hydrogen isotope effect of 2.4, becomes less prominent at higher pH values and is not detectable above pH 7. It is suggested that this process includes the formation of a covalent thiol-flavin C-4a derivative stabilized by protonation of the active site. In the presence of an excess of NADPH, the final product of the reaction is probably an NADPH complex of two-electron-reduced enzyme, but below pH 6 the final spectrum becomes less intense suggesting a partial formation of four-electron-reduced enzyme. The spectral changes observed above pH 7 are nearly independent of pH. The first measurable step (k = 48 s-1 at pH 9.5) is thought to include the formation of an NADP+ complex of two-electron-reduced enzyme, while the final step (k = 6.3 s-1 at pH 9.5) results in the above-mentioned NADPH complex with two-electron-reduced enzyme. A minimal kinetic scheme rationalizing the observed pH dependence of the reaction and the observed isotope effects is presented.     

The shikimate pathway. III. 3-dehydroquinate synthetase of E. coli. Mechanistic studies by kinetic isotope effect.

The conversion of 3-deoxy D-arabino heptulosonate 7-phosphate to 3-dehydroquinate by the 3-dehydroquinate synthetase from E. coli is characterized by a low but significant kinetic isotope effect for tritium carried in position-5 of DAHP, while no isotope effect was detectable for tritium in position-4. This effect was observed at different pH nad is interpreted as a result of theintermediary of a 5-ketonic form of the substrate, formed in a preliminary non limiting step during the enzymic cyclization reaction. A tentative scheme for the 3-DHQ synthetase reaction is proposed involving five steps: oxidation by NAD+ in position-5, phsophate elimination after enolization, reduction with precedently formed NADH and cyclization by attack of the 2-carbonyl by the C-7 methylene group.     

A secondary isotope effect in the lipoxygenase reaction

Fatty acids containing a prochiral tritium label have often been used in the study of enzymatic reactions which involve an obligatory step of hydrogen abstraction. In the lipoxygenase reaction, the primary isotope effect associated with this approach is detected as an isotopic enrichment of the substrate. Herein we characterize a previously unrecognized secondary isotope effect which changes the specific activity of both the substrate and product. The 12-lipoxygenase of human platelets removes the 10-LS hydrogen of arachidonic acid in the formation of 12-hydroperoxyeicosatetraenoic acid. We studied the specific activity changes associated with conversion of the enantiomerically labeled [10-DR-3H]arachidonic acid to 12-[10-3H]hydroxyeicosatetraenoic acid in aspirin-treated platelets. [3-14C]Arachidonic acid served as internal standard. The most pronounced change in 3H/14C ratio in the early stages of reaction was a 15-20% deficiency of tritium in the product. Later, the remaining arachidonate showed a marked increase in 3H/14C ratio. The changes in specific activity closely matched those predicted for a secondary isotope effect. Comparison of these data with the theoretical equations for a secondary isotope effect indicated the 10-DR-3H substrate reacted at about 84% of the rate of unlabeled molecules. Interestingly, this secondary isotope effect is similar in magnitude to the secondary isotope effect in autoxidation reactions, a finding compatible with a basic similarity in reaction mechanisms in enzymatic and non-enzymatic oxygenation of lipids.     

Enzyme mechanisms from molecular modeling and isotope effects

The application of kinetic isotope effects and molecular modeling to characterize three enzyme-catalyzed reactions is presented; the mechanism of the chloroacid dehalogenase catalyzed reaction is approached using chlorine kinetic isotope effects and solvent kinetic isotope effects. The pre-steady-state phase of the reaction catalyzed by methylmalonyl-CoA mutase is approached by different QM/MM schemes and the results are validated by comparison with the experimental value of the deuterium kinetic isotope effect. Finally, a procedure for improving QM/MM calculations is illustrated by analysis of the trihydroxynaphthalene reductase-catalyzed reaction.     

Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects

Mannitol dehydrogenases (MDH) are a family of Zn(2+)-independent long-chain alcohol dehydrogenases that catalyze the regiospecific NAD(+)-dependent oxidation of a secondary alcohol group in polyol substrates. pH and primary deuterium kinetic isotope effects on kinetic parameters for reaction of recombinant MDH from Pseudomonas fluorescens with D-mannitol have been measured in H(2)O and D(2)O at 25 degrees C and used to determine the relative timing of C-H and O-H bond cleavage steps during alcohol conversion. The enzymatic rates decreased at low pH; apparent pK values for log(k(cat)/K(mannitol)) and log k(cat) were 9.2 and 7.7 in H(2)O, respectively, and both were shifted by +0.4 pH units in D(2)O. Proton inventory plots for k(cat) and k(cat)/K(mannitol) were determined at pL 10.0 using protio or deuterio alcohol and were linear at the 95% confidence level. They revealed the independence of primary deuterium isotope effects on the atom fraction of deuterium in a mixed H(2)O-D(2)O solvent and yielded single-site transition-state fractionation factors of 0.43 +/- 0.05 and 0.47 +/- 0.01 for k(cat)/K(mannitol) and k(cat), respectively. (D)(k(cat)/K(mannitol)) was constant (1.80 +/- 0.20) in the pH range 6.0-9.5 and decreased at high pH to a limiting value of approximately 1. Measurement of (D)(k(cat)/K(fructose)) at pH 10.0 and 10.5 using NADH deuterium-labeled in the 4-pro-S position gave a value of 0.83, the equilibrium isotope effect on carbonyl group reduction. A mechanism of D-mannitol oxidation by MDH is supported by the data in which the partly rate-limiting transition state of hydride transfer is stabilized by a single solvation catalytic proton bridge. The chemical reaction involves a pH-dependent internal equilibrium which takes place prior to C-H bond cleavage and in which proton transfer from the reactive OH to the enzyme catalytic base may occur. Loss of a proton from the enzyme at high pH irreversibly locks the ternary complex with either alcohol or alkoxide bound in a conformation committed of undergoing NAD(+) reduction at a rate about 2.3-fold slower than the corresponding reaction rate of the protonated complex. Transient kinetic studies for D-mannitol oxidation at pH(D) 10.0 showed that the solvent isotope effect on steady-state turnover originates from a net rate constant of NADH release that is approximately 85% rate-limiting for k(cat) and 2-fold smaller in D(2)O than in H(2)O.     

Use of isotope effects to elucidate enzyme mechanisms

The chemical bond breaking steps are normally not rate limiting for enzymatic reactions. However, comparison of deuterium and tritium isotope effects on the same reaction, especially when coupled with 13C isotope effects for the same step measured with deuterated as well as unlabeled substrates, allows calculation of the intrinsic isotope effects on the bond breaking steps and thus a determination of the commitments to catalysis for the reactants. The variation in observed isotope effects as a function of reactant concentration can be used to determine kinetic mechanisms, while the pH variation of isotope effects can determine the stickiness of the reactants and which portions of the reactant mechanism are pH dependent. Finally the size of primary and secondary intrinsic isotope effects can be used to determine transition state structure.     

The pH dependence of the kinetic parameters of ketol acid reductoisomerase indicates a proton shuttle mechanism for alkyl migration.

The enzyme ketol acid reductoisomerase catalyzes the second common reaction in the biosynthesis of the branched chain amino acids. The reaction is complex as an alkyl migration and a ketone reduction apparently occur as separate steps during the conversion of acetolactate to 2,3-dihydroxy-3-methylbutyrate. This paper reports on the pH dependence of the kinetic parameters of the enzyme. The pH variation of log(V/K) for acetolactate was fit to an equation describing a bell-shaped curve, indicating an acid and a base catalyst for the reaction. In the reverse direction, V/K for 2,3-dihydroxy-3-methylbutyrate is constant over the pH range 8 to 10 and decreases below pH 8 with the ionization of two catalytic groups. The pH dependence of the V/K values for reduction of the kinetically competent intermediate and analogs of this intermediate are also described by a bell-shaped curve. The pH dependence of the V/K for alkyl migration of this intermediate indicates a single base catalyst for this reaction. We observe no deuterium kinetic isotope effect on V or V/K for the reaction of acetolactate at any pH. We observe a pH-dependent kinetic isotope effect on V/K for the reduction of the intermediate, the magnitude of which is metal ion dependent. Larger KIE's are observed in the presence of Mn2+ as opposed to Mg2+. In the reverse reaction there is a pH-dependent kinetic isotope effect on V/K. Based on the pH dependence of the kinetic parameters and the kinetic isotope effects, we propose a base-catalyzed proton shuttle mechanism for the alkyl migration reaction followed by an acid-assisted ketone reduction by NADPH.     

Alpha-secondary tritium isotope effects in the aqueous hydrolysis of glycopyranosides of N-acetyl-beta-D-glucosamine

Secondary tritium isotope effects were used to study the aqueous hydrolysis of a series of α- and β-glycopyranosides of N-acetyl-d-glucosamine. The magnitude of the secondary tritium isotope effects, and their dependence on the structure of the aglycone, are compatible with the carbonium ion mechanism suggested for the specific acid catalysis of these compounds by Piszkiewicz and Bruice (1–3). The secondary tritium isotope effect determined for the spontaneous hydrolysis of the p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside is not consistent with an intramolecular, nucleophilic displacement mechanism. A mechanism involving the equilibrium formation of a carbonium ion-anion pair is proposed. The relevance of these model studies to hydrolysis of oligosaccharides of N-acetyl-d-glucosamine by lysozyme is discussed.