Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase.

Tritiated adenosylcobalamin, labeled at the exchangeable position, has been used to investigate the partitioning of tritium between substrate and product in the reaction catalyzed by glutamate mutase. The isotope partitions between glutamate and methylaspartate in nearly 1:1 ratio, regardless of the direction in which the overall reaction is proceeding. This is consistent with a free-energy profile in which the interconversion of the intermediate glutamyl and methylaspartyl radicals is rapid relative to the transfer of tritium from 5'-deoxyadenosine to either substrate or product. Initial velocity measurements have been used to measure the tritium isotope effects for the transfer of tritium from adenosylcobalamin to product in each direction. The isotope effect is 21 for the formation of glutamate and 19 for the formation of methylasparate. The large magnitude of these isotope effects makes it likely that the rate-determining step may be altered by the substitution of tritium for hydrogen in the reaction. The results of these experiments are compared with previous isotope effect measurements made on other adenosylcobalamin-dependent enzymes.     

Protection of radical intermediates at the active site of adenosylcobalamin-dependent methylmalonyl-CoA mutase

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.     

Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. The reaction of (2R)-methylmalonyl-CoA in tritiated water.

The reaction catalysed by methylmalonyl-CoA epimerase from Propionibacterium shermanii was studied in tritiated water, in the direction with (2R)-methylmalonyl-CoA as substrate, under 'irreversible' conditions. After partial reaction, even when most of the substrate had been converted into product (isolated as propionyl-CoA) essentially no solvent tritium appeared in residual (2R)-methylmalonyl-CoA. The product, however, did contain tritium, and the specific radioactivity of the (2S)-epimer was deduced to be 0.33 times that of the solvent. These results provide further support for the mechanism proposed for the epimerase-catalysed reaction in the accompanying paper [Leadlay & Fuller (1983) Biochem. J. 213, 635-642], in which two enzyme bases act respectively as proton donor and acceptor. The observed low discrimination against solvent tritium entering the product can be accounted for by a mechanism in which the release of product is slow, and the re-protonation step on the enzyme is reversible, without leading to isotopic exchange with the solvent.     

The reaction of the substrate analog 2-ketoglutarate with adenosylcobalamin-dependent glutamate mutase

Glutamate mutase is one of several adenosylcobalamin-dependent enzymes that catalyze unusual rearrangements that proceed through a mechanism involving free radical intermediates. The enzyme exhibits remarkable specificity, and so far no molecules other than L-glutamate and L-threo-3-methylaspartate have been found to be substrates. Here we describe the reaction of glutamate mutase with the substrate analog, 2-ketoglutarate. Binding of 2-ketoglutarate (or its hydrate) to the holoenzyme elicits a change in the UV-visible spectrum consistent with the formation of cob(II)alamin on the enzyme. 2-ketoglutarate undergoes rapid exchange of tritium between the 5'-position of the coenzyme and C-4 of 2-ketoglutarate, consistent with the formation of a 2-ketoglutaryl radical analogous to that formed with glutamate. Under aerobic conditions this leads to the slow inactivation of the enzyme, presumably through reaction of free radical species with oxygen. Despite the formation of a substrate-like radical, no rearrangement of 2-ketoglutarate to 3-methyloxalacetate could be detected. The results indicate that formation of the C-4 radical of 2-ketoglutarate is a facile process but that it does not undergo further reactions, suggesting that this may be a useful substrate analog with which to investigate the mechanism of coenzyme homolysis.     

Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from [1(R)-3H]dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the "exchange-conversion" experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gamma-butyrobetaine hydroxylase: stereochemical course of the hydroxylation reaction

The stereochemical course of the aliphatic hydroxylation of gamma-butyrobetaine by calf liver and by Pseudomonas sp AK1 gamma-butyrobetaine hydroxylases has been determined. With [3(RS)-3-3H]-gamma-butyrobetaine or [3(R)-3-3H]-gamma-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [3(S)-3-3H]-gamma-butyrobetaine, only a negligible release of tritium to the aqueous medium was observed. Indeed, on hydroxylation of [3(S)-3-2H]-gamma-butyrobetaine by either the calf liver or bacterial hydroxylase, the isolated product L-carnitine was found to have retained all of the deuterium initially present in the 3(S) position. Since the absolute configuration of the product L-carnitine has been determined to be R, such results are only compatible with a hydroxylation reaction that proceeded with retention of configuration. With [methyl-14C,3(R)-3-3H]-gamma-butyrobetaine as substrate for the calf liver hydroxylase, the percentage of tritium retained in the [methyl-14C]-L-carnitine product was determined as a function of percent reaction. The results of these studies indicated that pro-R hydrogen atom abstraction exceeded 99.9%. Experiments using racemic [methyl-14C,3(RS)-3-3H]-gamma-butyrobetaine as substrate yielded similar results and additionally allowed us to estimate alpha-secondary tritium kinetic isotope effects of 1.10 and 1.31 for the bacterial and calf liver enzymes, respectively. These results are discussed within the context of the radical mechanism for gamma-butyrobetaine hydroxylase previously proposed [Blanchard, J. S., & Englard, S. (1983) Biochemistry 22, 5922], and the required topographical arrangement of enzymic oxidant and substrate is illustrated.     

Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. Studies with specifically tritiated (2R)-methylmalonyl-CoA as substrate.

(2R)-Methyl[2-3H]malonyl-CoA was used as the substrate for methylmalonyl-CoA epimerase from Propionibacterium shermanii, under conditions where the (2S)-methylmalonyl-CoA product was removed enzymically as fast as it was formed, and the fate of the label was monitored at different extents of reaction. Very little, if any, tritium is found attached to the C-2 position in the (2S)-epimer product (isolated as propionyl-CoA). Evidently, the hydrogen atom of the new C-H bond in the product is essentially solvent-derived. The rate of tritium release into the solvent is lower than the rate of product formation, and shows a primary kinetic tritium-isotope effect on kcat./Km of 2.3 +/- 0.1. The specific radioactivity of the remaining substrate rises slowly during the epimerase-catalysed reaction, and this provides an independent estimate of the primary kinetic tritium-isotope effect on kcat./Km of 1.6 +/- 0.5. These results, taken together, indicate that the mechanistic pathway of the epimerase-catalysed reaction resembles that established for proline racemase [Cardinale & Abeles, (1968) Biochemistry 7, 3970-3978], in which two enzyme bases are involved in catalysis. One base removes the proton from the substrate, the second provides the new proton, and there is no fast isotopic exchange between enzyme-bound intermediates and solvent protons.     

Mode of substrate interaction and energetics of carbon-oxygen bond formation of the dopamine beta-monooxygenase reaction.

Previous studies have shown that the dopamine beta-monooxygenase (DbetaM; E.C. 1.14.17.1)/1-(2-aminoethyl)-1,4-cyclohexadiene (CHDEA) reaction partitions between side chain and ring H-abstraction to produce the side-chain-hydroxylated product, 2-amino-1-(1, 4-cyclohexadienyl)ethanol, and the aromatized product, phenylethylamine, and that the two pathways do not crossover. [Wimalasena, K., and May, S. W. (1989) J. Am. Chem. Soc. 111, 2729-2731; Wimalasena, K., and Alliston, K. R. (1995) J. Am. Chem. Soc. 117, 1220-1224]. We now report that the ring H-abstraction pathway of the reaction further partitions to produce the ring hydroxylated product, CHDEA-6OH, and the aromatized product, PEA, at the carbon-oxygen bond formation step. The ring hydroxylation is shown to be stereospecific, exclusively producing the (S) product. The absolute stereospecificity of the ring and side-chain hydroxylations of the DbetaM/CHDEA reaction suggests that the side-chain pro-R hydrogen of the enzyme-bound substrate is close to perpendicular to the aromatic ring of the phenylethylamine substrate or cyclohexadiene ring of CHDEA. The relative activation energy parameters suggest that the partitioning of the ring H abstraction pathway between aromatized and ring hydroxylated products is due to the partitioning of the high-energy intermediates, the cyclohexadienyl radical and the Cu(II)-O(*) species, between carbon-oxygen bond formation and direct electron transfer. The relatively high activation enthalpic favorability and entropic unfavorability for the carbon-oxygen bond formation strongly suggest that the critical balancing of these two opposing forces is mandatory for the desired product formation.     

Magnitude of intrinsic isotope effects in the dopamine beta-monooxygenase reaction

Intrinsic primary hydrogen isotope effects (kH/kD) have been obtained for the carbon-hydrogen bond cleavage step catalyzed by dopamine beta-monooxygenase. Irreversibility of this step is inferred from the failure to observe back-exchange of tritium from TOH into substrate under conditions of dopamine turnover; this result cannot be due to solvent inaccessibility at the enzyme active site, since we will demonstrate [Ahn, N., & Klinman, J. P. (1983) Biochemistry (following paper in this issue)] that a solvent-derived proton or triton must be at the enzyme active site prior to substrate activation. As shown by Northrop [Northrop, D. B. (1975) Biochemistry 14, 2644], for enzymatic reactions in which the carbon-hydrogen bond cleavage step is irreversible, comparison of D(V/K) to T(V/K) allows an explicit solution for kH/kD. Employing a double-label tracer method, we have been able to measure deuterium isotope effects on Vmax/Km with high precision, D(V/K) = 2.756 +/- 0.054 at pH 6.0. The magnitude of the tritium isotope effect under comparable experimental conditions is T(V/K) = 6.079 +/- 0.220, yielding kH/kD = 9.4 +/- 1.3. This result was obtained in the presence of saturating concentrations of the anion activator fumarate. Elimination of fumarate from the reaction mixture leads to high observed values for isotope effects on Vmax/Km, together with an essentially invariant value for kH/kD = 10.9 +/- 1.9. Thus, the large disparity between isotope effects, plus or minus fumarate, cannot be accounted for by a change in kH/kD, and we conclude a role for fumarate in the modulation of the partitioning of enzyme-substrate complex between catalysis and substrate dissociation. On the basis of literature correlations of primary hydrogen isotope effects and the thermodynamic properties of hydrogen transfer reactions, the very large magnitude of kH/kD = 9.4-10.9 for dopamine beta-monooxygenase suggests an equilibrium constant not very far from unity for the carbon-hydrogen bond cleavage step. This feature, together with the failure to observe re-formation of dopamine from enzyme-bound intermediate or product and overall rate limitation of enzyme turnover by product release, leads us to propose a stepwise mechanism for norepinephrine formation from dopamine in which carbon-hydrogen bond cleavage is uncoupled from the oxygen insertion step.     

DNA alterations induced by the carbon-centered radical derived from the oxidation of 2-phenylethylhydrazine

The possible significance of carbon-centered radicals in hydrazine-induced carcinogenesis is explored by studies of the interaction between the 2-phenylethyl radical and DNA. The radical is efficiently generated during oxidation of phenelzine (2-phenylethylhydrazine) promoted by oxyhemoglobin or ferricyanide, as demonstrated by spin-trapping experiments and analysis of the reaction products. In the ferricyanide promoted oxidation, ethylbenzene formation accounts for about 40% of the initial drug concentration, from 5 to 100 mM phenelzine. By contrast, product formation in the presence of oxyhemoglobin depends on the enzyme concentration due to the fact that the prosthetic heme is destroyed during catalytic turnover. Covalent binding of the 2-phenylethyl radical to oxyhemoglobin is demonstrated by experiments with 2-[3H]phenelzine, where tritium incorporation to the protein is inhibited by the spin-trap, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. The 2-phenylethyl radical is also able to alkylate DNA as suggested by electrophoretic studies with plasmid DNA, and proved by experiments with 2-[3H]-phenelzine. The carbon-centered radical has a preference for attacking guanine residues as demonstrated by the use of sequencing techniques with 32P-DNA probes. The results indicate that the 2-phenylethyl radical is an important product of phenelzine oxidation and that this species can directly damage protein and DNA.     

Real-time monitoring of the oxalate decarboxylase reaction and probing hydron exchange in the product, formate, using fourier transform infrared spectroscopy

Oxalate decarboxylase converts oxalate to formate and carbon dioxide and uses dioxygen as a cofactor despite the reaction involving no net redox change. We have successfully used Fourier transform infrared spectroscopy to monitor in real time both substrate consumption and product formation for the first time. The assignment of the peaks was confirmed using [(13)C]oxalate as the substrate. The K(m) for oxalate determined using this assay was 3.8-fold lower than that estimated from a stopped assay. The infrared assay was also capable of distinguishing between oxalate decarboxylase and oxalate oxidase activity by the lack of formate being produced by the latter. In D(2)O, the product with oxalate decarboxylase was C-deuterio formate rather than formate, showing that the source of the hydron was solvent as expected. Large solvent deuterium kinetic isotope effects were observed on V(max) (7.1 +/- 0.3), K(m) for oxalate (3.9 +/- 0.9), and k(cat)/K(m) (1.8 +/- 0.4) indicative of a proton transfer event during a rate-limiting step. Semiempirical quantum mechanical calculations on the stability of formate-derived species gave an indication of the stability and nature of a likely enzyme-bound formyl radical catalytic intermediate. The capability of the enzyme to bind formate under conditions in which the enzyme is known to be active was determined by electron paramagnetic resonance. However, no enzyme-catalyzed exchange of the C-hydron of formate was observed using the infrared assay, suggesting that a formyl radical intermediate is not accessible in the reverse reaction. This restricts the formation of potentially harmful radical intermediates to the forward reaction.     

Analysis of ping-pong reaction mechanisms by positional isotope exchange. Application to galactose-1-phosphate uridyltransferase

A new positional isotope exchange method has been developed that can be used for the analysis of enzyme-catalyzed reactions which have ping-pong kinetic mechanisms. The technique can be used to measure the relative rates of ligand dissociation from enzyme-product complexes. Enzyme is incubated with the labeled substrate and an excess of the corresponding unlabeled product. The partitioning of the enzyme-product complex back toward free enzyme is determined from the rate of positional isotope exchange within the original labeled substrate. The partitioning of the enzyme-product complex forward toward free enzyme is determined from the rate of formation of totally unlabeled substrate. It has been shown that the ratio of the two rates provides a lower limit for the release of product from the enzyme-product complex. The technique has been applied to the reaction catalyzed by galactose-1-phosphate uridyltransferase. The lower limit for the release of glucose 1-phosphate from the uridyl-enzyme relative to the maximal velocity of the reverse reaction was determined to be 3.4 +/- 0.5.     

Importance of solute partitioning in biphasic oxidation of benzyl alcohol by free and immobilized whole cells of pichia pastoris

Using free and immobilized whole cells of Pichia pastoris, the biocatalytic oxidation of benzyl alcohol was investigated in different two-phase systems. This reaction was strongly influenced by both the substrate and product inhibitions, and the production rate of benzaldehyde in the aqueous system became maximum at the initial substrate concentration of ca. 29 g/L with the aldehyde formation less than 4 to 5 g/L even after a longer reaction period. The reaction rates in the two-liquid phase systems were predominantly determined by the partitioning behaviors of the substrate and product between the two phases rather than by enzyme deactivation by the organic solvents. In the two-liquid phase systems, consequently, the organic solvent acted as a reservior to reduce these inhibitory effects, and it was essential to select the organic solvent providing the optimal partitioning of the substrate into the aqueous phase as well as the preferential extraction of the product into the organic phase. The whole cells immobilized in a mixed matrix composed of silicone polymer [>50% (v/v)] and Ca alginate gel (<50%) worked well in the xylene and decane media, providing comparable activities with the free cells. The production rate of aldehyde was also influenced by the solute partitioning into the hydrophilic alginate phase where the cells existed. (c) 1994 John Wiley & Sons, Inc.     

Pre-steady-state kinetic investigation of intermediates in the reaction catalyzed by adenosylcobalamin-dependent glutamate mutase.

Glutamate mutase catalyzes the reversible isomerization of L-glutamate to L-threo-3-methylaspartate. Rapid quench experiments have been performed to measure apparent rate constants for several chemical steps in the reaction. The formation of substrate radicals when the enzyme was reacted with either glutamate or methylaspartate was examined by measuring the rate at which 5'-deoxyadenosine was formed, and shown to be sufficiently fast for this step to be kinetically competent. Furthermore, the apparent rate constant for 5'-deoxyadenosine formation was very similar to that measured previously for cleavage of the cobalt-carbon bond of adenosylcobalamin by the enzyme, providing further support for a mechanism in which homolysis of the coenzyme is coupled to hydrogen abstraction from the substrate. The pre-steady-state rates of methylaspartate and glutamate formation were also investigated. No burst phase was observed with either substrate, indicating that product release does not limit the rate of catalysis in either direction. For the conversion of glutamate to methylaspartate, a single chemical step appeared to dominate the overall rate, whereas in the reverse direction a lag phase was observed, suggesting the accumulation of an intermediate, tentatively ascribed to glycyl radical and acrylate. The rates of formation and decay of this intermediate were also sufficiently rapid for it to be kinetically competent. When combined with information from previous mechanistic studies, these results allow a qualitative free energy profile to constructed for the reaction catalyzed by glutamate mutase.     

Mass transfer induced interchange of the kinetic and thermodynamic control of enzymic peptide synthesis in biphasic water-organic systems

The α-chymotrypsin catalysed kinetically controlled peptide synthesis in water and in biphasic water-methyl iso-butyl ketone system was compared. Due to the substrate and product partitioning in the biphasic system an interchange of the reaction control was observed at high enzyme concentration. Under these conditions, the rate of mass transfer between the phases was the rate limiting step and the hydrolysis product concentration was found to have a transient maximum ≫ equilibrium value. In this case, most of the peptide was sythetized in a thermodynamically controlled process. In an aqueous one phase system, the peptide synthesis was kinetically controlled.     

Thermodynamic regulation of human short-chain acyl-CoA dehydrogenase by substrate and product binding

Human short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle for substrates with four and six carbons. Previous studies have shown that the act of substrate/product binding induces a large enzyme potential shift in acyl-CoA dehydrogenases. The objective of this work was to examine the thermodynamic regulation of this process through direct characterization of the electrochemical properties of hSCAD using spectroelectrochemical methodology. A large amount of substrate activation was observed in the enzymatic reaction of hSCAD (+33 mV), the greatest magnitude measured in any acyl-CoA dehydrogenase to date. To examine the role of the substrate as well as the product in electron transfer by hSCAD, a catalytic base mutation (E368Q) was constructed. The E368Q mutation inactivates the reductive and oxidative pathways such that the individual effects of substrate and product binding on the redox potential can be investigated. Optimal substrate (butyryl-CoA) was seen to shift the flavin redox potential slightly more positive (+38 mV) than did optimal product (crotonyl-CoA) (+31 mV), a finding opposite of that observed in another short-chain enzyme, bacterial SCAD. These results indicate that substrate redox activation occurs in hSCAD leading to a large enzyme midpoint potential shift. Substrate binding in hSCAD appears to make a larger contribution than does product to thermodynamic modulation.     

2'-Deoxy-2'-halonucleotides as alternate substrates and mechanism-based inactivators of Lactobacillus leichmannii ribonucleotide reductase

The interaction of the ribonucleoside-triphosphate reductase of Lactobacillus leichmannii with various 2'-halogenated ribo- and arabinonucleoside triphosphates has been investigated. All analogues examined acted as mechanism-based inactivators of the enzyme, producing base, triphosphate, and halide. In all cases, the inactive enzyme had developed the distinctive chromophore at 320 nm that is characteristic of enzyme inactivated by 2-methylene-3(2H)-furanone. The striking similarities between these results and those previously reported for the inactivation of this enzyme by 2'-chloro-2'-deoxyuridine triphosphate suggest a common reaction path for all 2'-halonucleotides. In the pyrimidine series, it was found that 2'-fluoro- and 2'-chloronucleotides partitioned between inactivation and formation of the normal reduction product 2'-deoxynucleotide. Normal reduction predominated with 2'-fluoronucleotides, whereas it was a minor pathway for 2'-chloro-2'-deoxyuridine triphosphate. With 2'-chloro-2'-deoxyuridine triphosphate, the relative partitioning between the two modes was pH dependent: the amount of 2'-deoxyuridine triphosphate formed increased 2.8-fold upon changing from pH 6.1 to pH 8.3. The ability of 2'-arabinohalonucleotides to inactivate ribonucleotide reductase and the variation of partitioning of the pyrimidine analogues with leaving group and reaction pH are consistent with our radical cation hypothesis and support the proposal that the difference between normal catalysis and inactivation is related to the protonation state of the reductase.     

Site-directed mutagenesis of glutamate 317 of bovine α-1,3Galactosyltransferase and its effect on enzyme activity: Implications for reaction mechanism

Bovine α1,3galactosyltransferase (α1,3GalT) transfers galactose from UDP-α-galactose to terminal β-linked galactosyl residues with retention of configuration of the incoming galactose residue. The epitope synthesized has been shown to be critical for xenotransplantation. According to a proposed double-displacement reaction mechanism, glutamate-317 (E317) is thought to be the catalytic nucleophile. The proposed catalytic role of E317 involves an initial nucleophilic attack with inversion of configuration and formation of a covalent sugar–enzyme intermediate between E317 and galactose from the donor substrate, followed by a second nucleophilic attack performed by the acceptor substrate with a second inversion of configuration. To determine whether E317 of α1,3GalT is critical for enzyme activity, site-directed mutagenesis was used to substitute alanine, aspartic acid, cysteine and histidine for E317. If the proposed reaction mechanism for the α1,3GalT enzyme is correct, E317D and E317H would produce active enzymes since they can act as nucleophiles. The non-conservative mutation E317A and conservative mutation E317C are predicted to produce inactive or very low activity enzymes since the E317A mutant cannot engage in a nucleophilic attack, and the E317C mutant would trap the galactose residue. The results obtained demonstrate that E317D and E317H mutants retained activity, albeit significantly less than the wild-type enzyme. Additionally, both E317A and E317C mutant also retained enzyme activity, suggesting that E317 is not the catalytic nucleophile proposed in the double-displacement mechanism. Therefore, either a different amino acid may act as the catalytic nucleophile or the reaction must proceed by a different mechanism.     

The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus: THE ROLE OF GLU232 IN CATALYSIS*

The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu²³² in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that k_red, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu²³² being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both k_red and k_red/K_d from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu²³² of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pK_a of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.     

Reaction mechanism of glutamate racemase, a pyridoxal phosphate-independent amino acid racemase.

Glutamate racemase of Pediococcus pentosaceus contained no cofactor, and was completely inactivated by a thiol reagent. The role of a cysteine residue in the enzyme reaction was studied by chemical modification. The modification of this cysteine residue resulted in a concomitant loss of activity. DL-Glutamate protected the enzyme from inactivation. The inactivated enzyme was reactivated by addition of dithiothreitol. The racemization in 2H2O showed an overshoot in the optical rotation of glutamate before the substrate was completely racemized. This indicates that the removal of alpha-hydrogen is the rate determining step. During the racemization of D- or L-glutamate in 3H2O, tritium was incorporated preferentially into the product. Glutamate is racemized by the enzyme probably through a two base mechanism.