Carbon isotope effects on the decarboxylation of carboxylic acids. Comparison of the lactate oxidase reaction and the degradation of pyruvate by H2O2.

The isotope effect at C-1 on the H2O2-catalysed decarboxylation of pyruvate (used as a model reaction for the enzymic reaction) increases between pH 3 and 10 from 1.0007 +/- 0.0004 to 1.0283 +/- 0.0014 (25 degrees C). This result indicates a change in the rate-determining step from formation of the tetrahedral intermediate to decarboxylation of this intermediate. Practically no isotope fractionation at C-1 (1.0011 +/- 0.0002, pH 6.0, 25 degrees C) is found in the lactate oxidase-catalysed decarboxylation of lactate, which is indicative for the existence of an irreversible O2-dependent step prior to the enzyme-catalysed decarboxylation. In addition, the result provides further evidence that dissociation of pyruvate and H2O2 from the enzyme can be excluded. The isotope effect at C-2 of lactate in the enzymic reaction (1.0048 +/- 0.0004) is attributed to the hydrogen transfer step from lactate to the coenzyme.     

Isotope effect studies of the pyruvate-dependent histidine decarboxylase from Lactobacillus 30a

The decarboxylation of histidine by the pyruvate-dependent histidine decarboxylase of Lactobacillus 30a shows a carbon isotope effect of k12/k13 = 1.0334 +/- 0.0005 and a nitrogen isotope effect k14/k15 = 0.9799 +/- 0.0006 at pH 4.8, 37 degrees C. The carbon isotope effect is slightly increased by deuteriation of the substrate and slightly decreased in D2O. The observed nitrogen isotope effect indicates that the imine nitrogen in the substrate-Schiff base intermediate complex is ordinarily protonated, and the pH dependence of the carbon isotope effect indicates that both protonated and unprotonated forms of this intermediate are capable of undergoing decarboxylation. As with the pyridoxal 5'-phosphate dependent enzyme, Schiff base formation and decarboxylation are jointly rate-limiting, with the intermediate histidine-pyruvate Schiff base showing a decarboxylation/Schiff base hydrolysis ratio of 0.5-1.0 at pH 4.8. The decarboxylation transition state is more reactant-like for the pyruvate-dependent enzyme than for the pyridoxal 5'-phosphate dependent enzyme. These studies find no particular energetic or catalytic advantage to the use of pyridoxal 5'-phosphate over covalently bound pyruvate in catalysis of the decarboxylation of histidine.     

Isotope effect studies of the role of metal ions in isocitrate dehydrogenase.

Pig heart NADP+-dependent isocitrate dehydrogenase requires a metal ion for activity. Under optimum conditions (pH 7.5, Mg2+ present), the carbon isotope effect is k12/k13 = 0.9989 +/- 0.0004 for the carboxyl carbon undergoing decarboxylation and hydrogen isotope effects are VmaxH/VmaxD = 1.09 +/- 0.04 and (Vmax/Km)H/(Vmax/Km)D = 0.76 +/- 0.12 with threo-D,L-[2-2H]isocitric acid. Deuterium isotope effects measured by the equilibrium perturbation technique under the same conditions are VH/VD = 1.20 for the forward reaction and 1.02 for the reverse reaction. Under these conditions the rate-determining step in the enzymatic reaction must be product release. Dissociation of isocitrate from the enzyme-isocitrate complex and the enzyme-NADP+ complex must be two or more orders of magnitude slower than the chemical steps. The catalytic activity of the enzyme is about tenfold lower in the presence of Ni2+ than in the presence of Mg2+. The carbon isotope effect in the presence of Ni2+ at pH 7.5 is k12/k13 = 1.0051 +/- 0.0012 and the hydrogen isotope effects are VmaxH/VmaxD = 0.98 +/- 0.07 and (Vmax/Km)H/(Vmax/Km)D = 1.11 +/- 0.14. Thus, the rate decrease caused by substitution of Ni2+ for Mg2+ must result from the effects of metal on substrate and product binding and dissociation, rather than effects of metal on catalysis. However, a more detailed analysis of the carbon isotope effects reveals that there is also a large metal effect on the rate of the decarboxylation step, consistent with the view that the carbonyl oxygen of the oxalosuccinate intermediate is coordinated to the metal during decarboxylation.     

C2-alpha-lactylthiamin diphosphate is an intermediate on the pathway of thiamin diphosphate-dependent pyruvate decarboxylation. Evidence on enzymes and models

Thiamin diphosphate (ThDP)-dependent decarboxylations are usually assumed to proceed by a series of covalent intermediates, the first one being the C2-trimethylthiazolium adduct with pyruvate, C2-alpha-lactylthiamin diphosphate (LThDP). Herein is addressed whether such an intermediate is kinetically competent with the enzymatic turnover numbers. In model studies it is shown that the first-order rate constant for decarboxylation can indeed exceed 50 s(-1) in tetrahydrofuran as solvent, approximately 10(3) times faster than achieved in previous model systems. When racemic LThDP was exposed to the E91D yeast pyruvate decarboxylase variant, or to the E1 subunit of the pyruvate dehydrogenase complex (PDHc-E1) from Escherichia coli, it was partitioned between reversion to pyruvate and decarboxylation. Under steady-state conditions, the rate of these reactions is severely limited by the release of ThDP from the enzyme. Under pre-steady-state conditions, the rate constant for decarboxylation on exposure of LThDP to the E1 subunit of the pyruvate dehydrogenase complex was 0.4 s(-1), still more than a 100-fold slower than the turnover number. Because these experiments include binding, decarboxylation, and oxidation (for detection purposes), this is a lower limit on the rate constant for decarboxylation. The reasons for this slow reaction most likely include a slow conformational change of the free LThDP to the V conformation enforced by the enzyme. Between the results from model studies and those from the two enzymes, it is proposed that LThDP is indeed on the decarboxylation pathway of the two enzymes studied, and once LThDP is bound the protein needs to provide little assistance other than a low polarity environment.     

Pyruvate decarboxylase is like acetolactate synthase (ILV2) and not like the pyruvate dehydrogenase E1 subunit

Protein sequences of pyruvate decarboxylase (PDC) derived from cloned yeast (Saccharomyces cerevisiae) and bacterial (Zymomonas mobilis) genes were compared with each other and with sequence databases. Extensive sequence similarities were found between them and with two others: cytochrome-linked pyruvate oxidase from Escherichia coli and acetolactate synthase (ilvI in E. coli; ILV2 gene in S. cerevisiae). All catalyse decarboxylation of pyruvate using thiamine pyrophosphate (TPP) as cofactor. General overall similarity suggests common ancestry for these enzymes. None of the sequences was similar to the E1 component of pyruvate dehydrogenase from E. coli which also decarboxylates pyruvate with the help of TPP.     

Tartrate dehydrogenase catalyzes the stepwise oxidative decarboxylation of D-malate with both NAD and thio-NAD

Tartrate dehydrogenase catalyzes the divalent metal ion- and NAD-dependent oxidative decarboxylation of D-malate to yield CO(2), pyruvate, and NADH. The enzyme also catalyzes the metal ion-dependent oxidation of (+)-tartrate to yield oxaloglycolate and NADH. pH-rate profiles and isotope effects were measured to probe the mechanism of this unique enzyme. Data suggest a general base mechanism with likely general acid catalysis in the oxidative decarboxylation of D-malate. Of interest, the mechanism of oxidative decarboxylation of D-malate is stepwise with NAD(+) or the more oxidizing thio-NAD(+). The mechanism does not become concerted with the latter as observed for the malic enzyme, which catalyzes the oxidative decarboxylation of L-malate [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096-2103]. It appears the change in mechanism observed with malic enzyme is specific to its transition state structure and not a generalized trait of metal ion- and NAD(P)-dependent beta-hydroxy acid oxidative decarboxylases. The V/K(malate) pH-rate profile decreases at low and high pH and exhibits pK(a) values of about 6.3 and 8.3, while that for V/K(tartrate) (measured from pH 7.5 to pH 9) exhibits a pK(a) of 8.6 on the basic side. A single pK(a) of 6.3 is observed on the acid side of the V(max) pH profile, but the pK(a) seen on the basic side of the V/K pH profiles is not observed in the V(max) pH profiles. Data suggest the requirement for a general base that accepts a proton from the 2-hydroxyl group of either substrate to facilitate hydride transfer. A second enzymatic group is also required protonated for optimum binding of substrates and may also function as a general acid to donate a proton to the enolpyruvate intermediate to form pyruvate. The (13)C isotope effect, measured on the decarboxylation of D-malate using NAD(+) as the dinucleotide substrate, decreases from a value of 1.0096 +/- 0.0006 with D-malate to 1.00787 +/- 0.00006 with D-malate-2-d, suggesting a stepwise mechanism for the oxidative decarboxylation of D-malate. Using thio-NAD(+) as the dinucleotide substrate the (13)C isotope effects are 1.0034 +/- 0.0007 and 1.0027 +/- 0.0002 with D-malate and D-malate-2-d, respectively.     

Nitrogen isotope effects on glutamate decarboxylase from Escherichia coli

The nitrogen isotope effect on the decarboxylation of glutamic acid by glutamate decarboxylase from Escherichia coli has been measured by comparison of the isotopic composition of the amino nitrogen of the product gamma-aminobutyric acid isolated after 10-20% reaction with that of the starting glutamic acid. At pH 4.7, 37 degrees C, the isotope effect is k14/k15 = 0.9855 +/- 0.0006 when compared to unprotonated glutamic acid. Interpretation of this result requires knowledge of the equilibrium nitrogen isotope effect for Schiff base formation. This equilibrium isotope effect is k14/k15 = 0.9824 for the formation of the unprotonated Schiff base between unprotonated valine and salicylaldehyde. Analysis of the nitrogen isotope effect on decarboxylation of glutamic acid and of the previously measured carbon isotope effect on this same reaction [O'Leary, M.H., Yamada, H., & Yapp, C.J. (1981) Biochemistry 20, 1476] shows that decarboxylation and Schiff base formation are jointly rate limiting. The enzyme-bound Schiff base between glutamate and pyridoxal 5'-phosphate partitions approximately 2:1 between decarboxylation and return to the starting state. The nitrogen isotope effect also reveals that the Schiff base nitrogen is protonated in this intermediate.     

Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k14/k15 = 0.9770 +/- 0.0021, a carbon isotope effect k12/k13 = 1.0308 +/- 0.0006, and a carbon isotope effect for L-[alpha-2H]histidine of 1.0333 +/- 0.0001 at pH 6.3, 37 degrees C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli [Abell, L. M., & O'Leary, M. H. (1988) Biochemistry 27, 3325], the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.     

Isotope effect studies of the chemical mechanism of nicotinamide adenine dinucleotide malic enzyme from Crassula

The 13C primary kinetic isotope effect on the decarboxylation of malate by nicotinamide adenine dinucleotide malic enzyme from Crassula argentea is 1.0199 +/- 0.0006 with proteo L-malate-2-H and 1.0162 +/- 0.0003 with malate-2-d. The primary deuterium isotope effect is 1.45 +/- 0.10 on V/K and 1.93 +/- 0.13 on Vmax. This indicates a stepwise conversion of malate to pyruvate and CO2 with hydride transfer preceding decarboxylation, thereby suggesting a discrete oxaloacetate intermediate. This is in agreement with the stepwise nature of the chemical mechanism of other malic enzymes despite the Crassula enzyme's inability to reduce or decarboxylate oxaloacetate. Differences in morphology and allosteric regulation between enzymes suggest specialization of the Crassula malic enzyme for the physiology of crassulacean acid metabolism while maintaining the catalytic events found in malic enzymes from animal sources.     

Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).     

Reaction mechanism for mammalian pyruvate dehydrogenase using natural lipoyl domain substrates

The pyruvate dehydrogenase (E1) component of the pyruvate dehydrogenase complex (PDC) catalyzes a two-step reaction. Recombinant production of substrate amounts of the lipoyl domains of the dihydrolipoyl transacetylase (E2) component of the mammalian PDC allowed kinetic characterization of the rapid physiological reaction catalyzed by E1. Using either the N-terminal (L1) or the internal (L2) lipoyl domain of E2 as a substrate, analyses of steady state kinetic data support a ping pong mechanism. Using standard E1 preparations, Michaelis constants (Km) were 52 +/- 14 microM for L1 and 24.8 +/- 3.8 microM for pyruvate and k(cat) was 26.3 s(-1). With less common, higher activity preparations of E1, the Km values were > or =160 microM for L1 and > or =35 microM for pyruvate and k(cat) was > or =70 s(-1). Similar results were found with the L2 domain. The best synthetic lipoylated-peptide (L2 residues 163-177) was a much poorer substrate (Km > or =15 mM, k(cat) approximately equals 5 s(-1); k(cat)/Km decreased >1,500-fold) than L1 or L2, but a far better substrate in the E1 reaction than free lipoamide (k(cat)/Km increased >500-fold). Each lipoate source was an effective substrate in the dihydrolipoyl dehydrogenase (E3) reaction, but E3 had a lower Km for the L2 domain than for lipoamide or the lipoylated peptides. In contrast to measurements with slow E1 model reactions that use artificial acceptors, we confirmed that the natural E1 reaction, using lipoyl domain acceptors, was completely inhibited (>99%) by phosphorylation of E1 and the phosphorylation strongly inhibited the reverse of the second step catalyzed by E1. The mechanisms by which phosphorylation interferes with E1 activity is interpreted based on accrued results and the location of phosphorylation sites mapped onto the 3-D structure of related alpha-keto acid dehydrogenases.     

A Secondary beta Deuterium Kinetic Isotope Effect in the Chorismate Synthase Reaction

Chorismate synthase (EC 4.6.1.4) is the shikimate pathway enzyme that catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The enzyme reaction is unusual because it involves a trans-1,4 elimination of the C-3 phosphate and the C-6 proR hydrogen and it has an absolute requirement for reduced flavin. Several mechanisms have been proposed to account for the cofactor requirement and stereochemistry of the reaction, including a radical mechanism. This paper describes the synthesis of [4-(2)H]EPSP and the observation of kinetic isotope effects using this substrate with both Neurospora crassa and Escherichia coli chorismate synthases. The magnitude of the effects were (D)(V) = 1.08 +/- 0.01 for the N. crassa enzyme and 1.10 +/- 0.02 on phosphate release under single-turnover conditions for the E. coli enzyme. The effects are best rationalised as substantial secondary beta isotope effects. It is most likely that the C(3)-O bond is cleaved first in a nonconcerted E1 or radical reaction mechanism. Although this study alone cannot rule out a concerted E2-type mechanism, the C(3)-O bond would have to be substantially more broken than the proR C(6)-H bond in a transition state of such a mechanism. Importantly, although the E. coli and N. crassa enzymes have different rate limiting steps, their catalytic mechanisms are most likely to be chemically identical. Copyright 2000 Academic Press.     

Carbon-13 and deuterium isotope effects on oxalacetate decarboxylation by pyruvate carboxylase

Deuterium and 13C isotope effects for the enzymic decarboxylation of oxalacetate showed that both deuterium- and 13C-sensitive steps in the reaction are partially rate limiting. A normal alpha-secondary effect of 1.2 per deuterium was calculated for the reaction in which pyruvate-d3 was the substrate, suggesting that the enolate of pyruvate was an intermediate in the reaction. The large normal alpha-secondary deuterium isotope effect of 1.7 when oxalacetate-d2 was the substrate suggests that the motions of the secondary hydrogens are coupled to that of the primary hydrogen during the protonation of the enolate of pyruvate. The reduction in the magnitude of the 13C isotope effect for the oxamate-dependent decarboxylation of oxalacetate from 1.0238 to 1.0155 when the reaction was performed in D2O (primary deuterum isotope effect = 2.1) clearly indicates that the transfer of the proton and carboxyl group between biotin and pyruvate does not occur via a single concerted reaction. Mechanisms in which biotin is activated to react with CO2 (prior to transfer of the proton on N-1) by bond formation between the sulfur and the ureido carbon, or in which the sequence of events is decarboxylation of oxalacetate, proton transfer from biotin to enolpyruvate, and carboxylation of enolbiotin, predict that the 13C isotope effect in D2O should be substantially lower than the observed value. A stepwise mechanism that does fit the data is one in which a proton is removed from biotin by a sulfhydryl group on the enzyme prior to carboxyl transfer, as long as the sulfhydryl group has an abnormally low pK.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid kinetic and isotopic studies on dialkylglycine decarboxylase

The two half-reactions of the pyridoxal 5'-phosphate (PLP)-dependent enzyme dialkylglycine decarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasic behavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observed in steady-state kinetics [Zhou, X., and Toney, M. D. (1998) Biochemistry 37, 5761--5769]. The half-reaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steady-state kinetic parameters. The fast phase maximal rate constant for the 2-aminoisobutyrate (AIB) decarboxylation half-reaction with the potassium form of DGD-PLP is 25 s(-1), while that for the transamination half-reaction between DGD-PMP and pyruvate is 75 s(-1). The maximal rate constant for the transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s(-1). The spectral data indicate that external aldimine formation with either AIB or L-alanine and DGD-PLP is a rapid equilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable to the quinonoid intermediate is not observed in the AIB decarboxylation half-reaction, but is observed in the dead-time of the stopped-flow in the L-alanine transamination half-reaction. The [1-(13)C]AIB kinetic isotope effect (KIE) on k(cat) for the steady-state reaction is 1.043 +/- 0.003, while a value of 1.042 +/- 0.009 was measured for the AIB half-reaction. The secondary KIE measured for the AIB decarboxylation half-reaction with [C4'-(2)H]PLP is 0.92 +/- 0.02. The primary [2-(2)H]-L-alanine KIE on the transamination half-reaction is unity. Small but significant solvent KIEs are observed on k(cat) and k(cat)/K(M) for both substrates, and the proton inventories are linear in each case. NMR measurements of C2--H washout vs product formation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. These results support a rate-limiting, concerted C alpha-decarboxylation/C4'-protonation mechanism for the AIB decarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate-limiting protonation to the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the two half-reactions are constructed.     

Analyzing the catalytic mechanism of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

In order to gain insight into the catalytic mechanism of Fe-type nitrile hydratases (NHase), the pH and temperature dependence of the kinetic parameters k cat, K m, and k cat/ K m along with the solvent isotope effect were examined for the Fe-type NHase from Comamonas testosteroni Ni1 ( CtNHase). CtNHase was found to exhibit a bell-shaped curve for plots of relative activity vs pH over pH values 4-10 for the hydration of acrylonitrile and was found to display maximal activity at pH approximately 7.2. Fits of these data provided a p K ES1 value of 6.1 +/- 0.1, a p K ES2 value of 9.1 +/- 0.2 ( k' cat = 10.1 +/- 0.3 s (-1)), a p K E1 value of 6.2 +/- 0.1, and a p K E2 value of 9.2 +/- 0.1 ( k' cat/ K' m of 2.0 +/- 0.2 s (-1) mM (-1)). Proton inventory studies indicate that two protons are transferred in the rate-limiting step of the reaction at pH 7.2. Since CtNHase is stable to 25 degrees C, an Arrhenius plot was constructed by plotting ln( k cat) vs 1/ T, providing an E a of 33.3 +/- 1.5 kJ/mol. Delta H degrees of ionization values were also determined, thus helping to identify the ionizing groups exhibiting the p K ES1 and p K ES2 values. Based on Delta H degrees ion data, p K ES1 is assigned to betaTyr68 while p K ES2 is assigned to betaArg52, betaArg157, or alphaSer116 (NHases are alpha 2beta 2 heterotetramers). Given the strong similarities in the kinetic data obtained for both Co- and Fe-type NHase enzymes, both types of NHase enzymes likely hydrate nitriles in a similar fashion.     

The roles of Tyr(91) and Lys(162) in general acid-base catalysis in the pigeon NADP+-dependent malic enzyme

The role of general acid-base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr(91) and Lys(162) residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in k(cat) values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the k(cat) values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(k(cat)) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pK(a) value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys(162) as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr(91) residue could form a hydrogen bond with Lys(162) to act as a catalytic dyad that contributes a proton to complete the enol-keto tautomerization.     

Solvent isotope effect on the reaction catalysed by the pyruvate dehydrogenase complex from Escherichia coli

The pyruvate dehydrogenase from Escherichia coli showed a primary kinetic isotope effect when its overall reaction or the partial reaction of the pyruvate dehydrogenase component were tested in deuterium oxide. The Michaelis constants for pyruvate were nearly unchanged, but the maximum velocities in water and deuterium oxide differed, their ratio being DV = 1.7 for the overall reaction and DV = 2.1 for the E1p reaction. The pH profile and, accordingly, the delta pK1 and delta pK2 values were shifted by 0.6 units to higher pL values. A linear proton inventory curve was obtained when varying the atom fractions of protons relative to deuterons from 100 to 0%. This is an indication for a single proton transfer. It is proposed that this relatively weak primary isotope effect may be caused by the protonation of the N1' nitrogen at the pyrimidine ring of the cofactor by an adjacent glutamate residue. The proton of its carboxylic group exchanges very fast with deuterons of the solvent.     

Isotope effects on binding of NAD+ to lactate dehydrogenase

The isotope effect on binding [4-2H]NAD+ and [4-3H]NAD+ to lactate dehydrogenase has been shown to be 1.10 +/- 0.03 by whole molecule isotope ratio mass spectrometry and 1.085 +/- 0.01 by 3H/14C scintillation counting. These values demonstrate that specific interactions of the nicotinamide ring with the enzyme make the C-H bond at C-4 less stiff in the binary complex.     

Mechanisms of enzymatic and acid-catalyzed decarboxylations of prephenate

The prephenate dehydrogenase activity of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase from Escherichia coli catalyzes the oxidative decarboxylation of both prephenate and deoxoprephenate, which lacks the keto group in the side chain (V 78% and V/K 18% those of prephenate). Hydride transfer is to the B side of NAD, and the acetylpyridine and pyridinecarboxaldehyde analogues of NAD have V/K values 40 and 9% and V values 107 and 13% those of NAD. Since the 13C isotope effect on the decarboxylation is 1.0103 with deuterated and 1.0033 with unlabeled deoxoprephenate (the deuterium isotope effect on V/K is 2.34), the mechanism is concerted, and if CO2 has no reverse commitment, the intrinsic 13C and deuterium isotope effects are 1.0155 (corresponding to a very early transition state for C-C bond cleavage) and 7.3, and the forward commitment is 3.7. With deoxodihydroprephenate (lacking one double bond in the ring), oxidation occurs without decarboxylation, and one enantiomer has a V/K value 23-fold higher than the other (deuterium isotope effects are 3.6 and 4.1 for fast and slow isomers; V for the fast isomer is 5% and V/K 0.7% those of prephenate). The fully saturated analogue of deoxoprephenate is a very slow substrate (V 0.07% and V/K approximately 10(-5%) those of prephenate). pH profiles show a group with pK = 8.3 that must be protonated for substrate binding and a catalytic group with pK = 6.5 that is a cationic acid (likely histidine). This group facilitates hydride transfer by beginning to accept the proton from the 4-hydroxyl group of prephenate prior to the beginning of C-C cleavage (or fully accepting it in the oxidation of the analogues with only one double bond or none in the ring). In contrast with the enzymatic reaction, the acid-catalyzed decarboxylation of prephenate and deoxoprephenate (t1/2 of 3.7 min at low pH) is a stepwise reaction with a carbonium ion intermediate, since 18O is incorporated into substrate and its epi isomer during reaction in H218O. pH profiles show that the hydroxyl group must be protonated and the carboxyl (pK approximately 4.2) ionized for carbonium ion formation. The carbonium ion formed from prephenate decarboxylates 1.75 times faster than it reacts with water (giving 1.8 times as much prephenate as epi isomer). The observed 13C isotope effect of 1.0082 thus corresponds to an intrinsic isotope effect of 1.023, indicating an early transition state for the decarboxylation step. epi-Prephenate is at least 20 times more stable to acid than prephenate because it exists largely as an internal hemiketal.(ABSTRACT TRUNCATED AT 400 WORDS)