Roles of his205, his296, his303 and Asp259 in catalysis by NAD+-specific D-lactate dehydrogenase.

The role of three histidine residues (His205, His296 and His303) and Asp259, important for the catalysis of NAD+-specific D-lactate dehydrogenase, was investigated using site-directed mutagenesis. None of these residues is presumed to be involved in coenzyme binding because Km for NADH remained essentially unchanged for all the mutant enzymes. Replacement of His205 with lysine resulted in a 125-fold reduction in kcat and a slight lowering of the Km value for pyruvate. D259N mutant showed a 56-fold reduction in kcat and a fivefold lowering of Km. The enzymatic activity profile shifted towards acidic pH by approximately 2 units. The H303K mutation produced no significant change in kcat values, although Km for pyruvate increased fourfold. Substitution of His296 with lysine produced no significant change in kcat values or in Km for substrate. The results obtained suggest that His205 and Asp259 play an important role in catalysis, whereas His303 does not. This corroborates structural information available for some members of the D-specific dehydrogenases family. The catalytic His296, proposed from structural studies to be the active site acid/base catalyst, is not invariant. Its function can be accomplished by lysine and this has significant implications for the enzymatic mechanism.     

Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli.

NAD(+)-dependent D-lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N-terminal sequence and amino acid residues 220-215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267, 8499-8513], a 0.6-kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the D-lactate dehydrogenase gene. Both plasmids overexpressed D-lactate dehydrogenase (greater than 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticus D-lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of D-2-hydroxy-acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60-66]. The physicochemical and catalytic properties of recombinant D-lactate dehydrogenase were identical to those of the wild-type enzyme, e.g. alpha 2 dimeric subunit structure, isoelectric pH, Km and Kcat for pyruvate and other 2-oxo-acid substrates. The kinetic profiles of 2-oxo-acid substrates showed some marked differences from that of L-lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity.     

Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity

The function of aspartic acid residue 101 in the active site of Escherichia coli alkaline phosphatase was investigated by site-specific mutagenesis. A mutant version of alkaline phosphatase was constructed with alanine in place of aspartic acid at position 101. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, pH 8.0, both the kcat and the Km for the mutant enzyme increase by approximately 2-fold, resulting in almost no change in the kcat/Km ratio. Under conditions of no external phosphate acceptor and pH 8.0, both the kcat and the Km for the mutant enzyme decrease by approximately 2-fold, again resulting in almost no change in the kcat/Km ratio. The kcat for the hydrolysis of 4-methyl-umbelliferyl phosphate and p-nitrophenyl phosphate are nearly identical for both the wild-type and mutant enzymes, as is the Ki for inorganic phosphate. The replacement of aspartic acid 101 by alanine does have a significant effect on the activity of the enzyme as a function of pH, especially in the presence of a phosphate acceptor. At pH 9.4 the mutant enzyme exhibits 3-fold higher activity than the wild-type. The mutant enzyme also exhibits a substantial decrease in thermal stability: it is half inactivated by treatment at 49 degrees C for 15 min compared to 71 degrees C for the wild-type enzyme. The data reported here suggest that this amino acid substitution alters the rates of steps after the formation of the phospho-enzyme intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase

We have explored the substrate protonation mechanism of Escherichia coli dihydrofolate reductase by changing the location of the proton donor. A double mutant was constructed in which the proton donor of the wild-type enzyme, aspartic acid-27, has been changed to serine and simultaneously an alternative proton donor, glutamic acid, has replaced threonine at position 113. The active site of the resulting variant enzyme molecule should therefore somewhat resemble that proposed for the R67 plasmid-encoded dihydrofolate reductase [Matthews, D. A., Smith, S. L., Baccanari, D. P., Burchall, J. J., Oatley, S. J., & Kraut, J. (1986) Biochemistry 25, 4194]. At pH 7, the double-mutant enzyme has a 3-fold greater kcat and an unchanged Km(dihydrofolate) as compared with the single-mutant Asp-27----Ser enzyme described previously [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science (Washington, D.C.) 231, 1123]. Additionally, its activity vs pH profiles together with observed deuterium isotope effects, suggest that catalysis depends on an acidic group with a pKa of 8. It is concluded that the dihydropteridine ring of a bound substrate molecule can indeed be protonated by a glutamic acid side chain at position 113 (instead of an aspartic acid side chain at position 27), but with greatly decreased efficiency: at pH 7, the double mutant still has a 25-fold lower kcat (1.2 s-1) and a 2900-fold lower kcat/km(dihydrofolate) (8.6 X 10(3) s-1 M-1) than the wild-type enzyme.     

Molecular mechanism of VanHst, an alpha-ketoacid dehydrogenase required for glycopeptide antibiotic resistance from a glycopeptide producing organism.

The vancomycin resistance enzyme VanH is an alpha-ketoacid dehydrogenase that stereospecifically reduces pyruvate to D-lactate, which is required for the synthesis of the depsipeptide D-alanine-D-lactate. This compound then forms an integral part of the bacterial cell wall replacing the vancomycin target dipeptide D-alanine-D-alanine, thus the presence of VanH is essential for glycopeptide resistance. In this work, the VanH homologue from the glycopeptide antibiotic producing organism Streptomyces toyocaensis NRRL 15009, VanHst, has been overexpressed in Escherichia coli and purified, and its substrate specificity and mechanism were probed by steady-state kinetic methods and site-directed mutagenesis. The enzyme is highly efficient at pyruvate reduction with kcat/Km = 1.3 x 10(5) M-1 s-1 and has a more restricted alpha-ketoacid substrate specificity than VanH from vancomycin resistant enterococci (VRE). Conversely, VanHst shows no preference between NADH and NADPH while VanH from VRE prefers NADPH. The kinetic mechanism for VanHst was determined using product and dead-end inhibitors to be ordered BiBi with NADH binding first followed by pyruvate and products leaving in the order D-lactate, NAD+. Site-directed mutagenesis indicated that Arg237 plays a role in pyruvate binding and catalysis and that His298 is a candidate for an active-site proton donor. Glu266, which has been suggested to modulate the pKa of the catalytic His in other D-lactate dehydrogenases, was found to fulfill a similar role in VanHst, lowering a pKa value of kcat/Km nearly 2 units. These results now provide the framework for additional structure and inhibitor design work on the VanH family of antibiotic resistance enzymes.     

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.     

Functional characterization and crystal structure of the C215D mutant of protein-tyrosine phosphatase-1B

We have characterized the C215D active-site mutant of protein-tyrosine phosphatase-1B (PTP-1B) and solved the crystal structure of the catalytic domain of the apoenzyme to a resolution of 1.6 A. The mutant enzyme displayed maximal catalytic activity at pH approximately 4.5, which is significantly lower than the pH optimum of 6 for wild-type PTP-1B. Although both forms of the enzyme exhibited identical Km values for hydrolysis of p-nitrophenyl phosphate at pH 4.5 and 6, the kcat values of C215D were approximately 70- and approximately 7000-fold lower than those of wild-type PTP-1B, respectively. Arrhenius plots revealed that the mutant and wild-type enzymes displayed activation energies of 61 +/- 1 and 18 +/- 2 kJ/mol, respectively, at their pH optima. Unlike wild-type PTP-1B, C215D-mediated p-nitrophenyl phosphate hydrolysis was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane, suggesting a direct involvement of Asp215 in catalysis. Increasing solvent microviscosity with sucrose (up to 40% (w/v)) caused a significant decrease in kcat/Km of the wild-type enzyme, but did not alter the catalytic efficiency of the mutant protein. Structurally, the apoenzyme was identical to wild-type PTP-1B, aside from the flexible WPD loop region, which was in both "open" and "closed" conformations. At physiological pH, the C215D mutant of PTP-1B should be an effective substrate-trapping mutant that can be used to identify cellular substrates of PTP-1B. In addition, because of its insensitivity to oxidation, this mutant may be used for screening fermentation broth and other natural products to identify inhibitors of PTP-1B.     

Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3 x 10(4) times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km of H250N mutant was reduced by a factor of 2.5 x 10(4)-fold as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.     

Identification of the catalytic residues in family 52 glycoside hydrolase,a beta-xylosidase from Geobacillus stearothermophilus T-6

beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa). The mutant exhibited 103-fold reduction in activity, and the Br?nsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2.     

Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site.

The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.     

Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B

Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.     

Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site.

Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.     

Mutagenic and enzymological studies of the hydratase and isomerase activities of 2-enoyl-CoA hydratase-1

Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-enoyl-CoA hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-enoyl-CoA hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Delta3-Delta2-enoyl-CoA isomerase activity of mECH-1, a new assay using 2-enoyl-CoA hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively. The data are in line with the proposal that Glu164 acts as a protic amino acid residue for both the hydration and the isomerization reaction. The structural factors favoring the hydratase over the isomerase reaction have been addressed by investigating the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. The Gln162 side chain is hydrogen bonded to the Glu164 side chain; nevertheless, these mutants have enzymatic properties similar to that of the wild type, indicating that catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on the interactions with the Gln162 side chain.     

A strong carboxylate-arginine interaction is important in substrate orientation and recognition in lactate dehydrogenase

Using site-directed mutagenesis, Arginine-171 at the substrate-binding site of Bacillus stearothermophilus, lactate dehydrogenase has been replaced by lysine. In the closely homologous eukaryotic lactate dehydrogenase, this residue binds the carboxylate group of the substrate by forming a planar bifurcated bond. The mutation diminishes the binding energy of pyruvate, alpha-ketobutyrate and alpha-ketovalerate (measured by kcat/Km) by the same amount (about 6 kcal/mol). For each additional methylene group on the substrate, there is a loss of about 1.5 kcal/mol of binding energy in both mutant and wild-type enzymes. From these parallel trends in the two forms of enzyme, we infer that the mode of productive substrate binding is identical in each, the only difference being the loss of a strong carboxylate-guanidinium interaction in the mutant. In contrast to this simple pattern in kcat/Km, the Km alone increases with substrate-size in the wild-type enzyme, but decreases in the mutant. These results can be most simply explained by the occurrence of relatively tight unproductive enzyme-substrate complexes in the mutant enzyme as the substrate alkyl chain is extended. This does not occur in the wild-type enzyme, because the strong orienting effect of Arg-171 maximizes the frequency of substrates binding in the correct alignment.     

Identification of the principal catalytically important acidic residue of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Kinetic analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has implicated a glutamate or aspartate residue in (i) formation of mevaldate thiohemiacetal by proton transfer to the carbonyl oxygen of mevaldate and (ii) enhanced ionization of CoASH by the resulting enzyme carboxylate anion, facilitating attack by CoAS- on the carbonyl carbon of mevaldate (Veloso, D., Cleland, W. W., and Porter, J. W. (1981) Biochemistry 81, 887-894). Although neither the identity of this acidic residue nor its location is known, the catalytic domains of 11 sequenced HMG-CoA reductases contain only 3 conserved acidic residues. For HMG-CoA reductase of Pseudomonas mevalonii, these residues are Glu52, Glu83, and Asp183. To identify the acidic residue that functions in catalysis, we generated mutants having alterations in these residues. The mutant proteins were expressed, purified, and characterized. Mutational alteration of residues Glu52 or Asp183 of P. mevalonii HMG-CoA reductase yielded enzymes with significant, but in some cases reduced, activity (Vmax = 100% Asp183----Ala, 65% Asp183----Asn, and 15% Glu52----Gln of wild-type activity, respectively). Although the activity of mutant enzymes Glu52----Gln and Asp183----Ala was undetectable under standard assay conditions, their Km values for substrates were 4-300-fold higher than those for wild-type enzyme. Km values for wild-type enzyme and for mutant enzymes Glu52----Gln and Asp183----Ala were, respectively: 0.41, 73, and 120 mM [R,S)-mevalonate); 0.080, 4.4, and 2.0 mM (coenzyme A); and 0.26, 4.4, and 1.0 mM (NAD+). By these criteria, neither Glu52 nor Asp183 is the acidic catalytic residue although each may function in substrate recognition. During chromatography on coenzyme A agarose or HMG-CoA agarose, mutant enzymes Asp183----Asn and Glu83----Gln behaved like wild-type enzyme. By contrast, and in support of a role for these residues in substrate recognition, mutant enzymes Glu52----Gln and Asp183----Ala exhibited impaired ability to bind to either support. Despite displaying Km values for substrates and chromatographic behavior on substrate affinity supports comparable to wild-type enzyme, only mutant enzyme Glu83----Gln was essentially inactive under all conditions studied (Vmax = 0.2% that of wild-type enzyme). Glutamate residue 83 of P. mevalonii HMG-CoA reductase, and consequently the glutamate of the consensus Pro-Met-Ala-Thr-Thr-Glu-Gly-Cys-Leu-Val-Ala motif of the catalytic domains of eukaryotic HMG-CoA reductases, is judged to be the acidic residue functional in catalysis.     

枯草杆菌及大肠杆菌异柠檬酸脱氢酶的酶学性质研究

对枯草杆菌异柠檬酸脱氢酶（BsIDH）、大肠杆菌异柠檬酸脱氢酶（EcIDH）和大肠杆菌异柠檬酸脱氢酶的突变体酶（EmIDH）进行了纯化和酶学性质鉴定。BsIDH和EcIDH对辅酶NADP^＋的特异性与NAD^＋相比,分别是NAD^＋的1330倍和3890倍。而EmIDH对NAD^＋的特异性与NADP^＋相比,是NADP^＋的122倍。因此BsIDH和EcIDH是NADP^＋依赖性异柠檬酸脱氢酶,而EmIDH的辅酶特异性已转换为NAD^＋依赖性。EcIDH、BsIDH和EmIDH对底物异柠檬酸的Km值分别为67.4 μmol/L、60.6 μmol/L和105.6 μmol/L。BsIDH和EcIDH的最适反应pH分别为8.2和8.0,EmIDH的最适pH为7.0。BsIDH和EmIDH的最适反应温度是45℃,EcIDH的最适温度为43℃。三种IDH的活性依赖于不同的二价金属离子的存在,Mn^2＋ 、Mg^2＋存在时酶活性最强,Cu^2＋ 、Ca^2＋ 、Zn^2＋和Ni2＋强烈抑制酶的活性。系统的酶学性质研究为深入认识IDH的催化与调节机制提供了更多依据。     

Role of Glu312 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase

Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Nepsilon2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 value similar to that of the wild-type enzyme but kcat and kcat/Km values that were 230 and 35 times lower, respectively, than in the wild-type enzyme. These data are consistent with the spatial location of the negative charge on residue 312 being important for the oxidation of the alcohol substrate. Solvent viscosity and substrate kinetic isotope effects suggest the presence of an internal equilibrium in the Glu312Asp enzyme prior to the hydride transfer reaction. Altogether, the crystallographic and mechanistic data suggest that Glu312 is important for binding and positioning of the substrate in the active site of choline oxidase.     

Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin

Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.     

Identification of the catalytically important histidine of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We identify His381 of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase as the basic residue functional in catalysis. The catalytic domain of 20 HMG-CoA reductases contains a single conserved histidine (His381 of the P. mevalonii enzyme). Diethyl pyrocarbonate inactivated the P. mevalonii enzyme, and hydroxylamine partially restored activity. We changed His381 to alanine, lysine, asparagine, and glutamine. The mutant proteins were overexpressed, purified to homogeneity, and characterized. His381 mutant enzymes were not inactivated by diethyl pyrocarbonate. All four mutant enzymes exhibited wild-type crystal morphology and chromatographed on substrate affinity supports like wild-type enzyme. The mutant enzymes had low catalytic activity (Vmax 0.06-0.5% that of wild-type enzyme), but Km values approximated those for wild-type enzyme. For wild-type enzyme and mutant enzymes H381A, H381N, and H381Q, Km values at pH 8.1 were 0.45, 0.27, 3.7, and 0.71 mM [(R,S)-mevalonate]; 0.05, 0.03, 0.20, and 0.11 mM [coenzyme A]; 0.22, 0.14, 0.81, and 0.62 mM [NAD+]. Km values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM [(R,S)-mevalonate]; 0.24 and 0.50 mM [coenzyme A]; 0.15 and 1.23 mM [NAD+]. Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK1 and by 0.5-1.3 pH units for pK2). For mutant enzyme H381K, the pK1 of 10.2 is consistent with lysine acting as a general base at high pH. His381 of P. mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.     

Enzymatic effects of a lysine-to-glutamine mutation in the ATP-binding consensus sequence in the RecD subunit of the RecBCD enzyme from Escherichia coli.

The RecBCD-K177Q enzyme has a lysine-to-glutamine mutation in the putative ATP-binding sequence of the RecD protein (Korangy, F., and Julin, D.A. (1992) J. Biol. Chem. 267, 1727-1732). We have compared the enzymatic properties of the RecBCD-K177Q enzyme with those of the wild-type RecBCD enzyme from Escherichia coli. The purified RecBCD-K177Q enzyme has ATP-dependent nuclease activity on double-stranded or denatured DNA which is reduced (4-14-fold less) compared with the wild type. The kcat and Km(ATP) for ATP hydrolysis stimulated by double-stranded DNA are both reduced in RecBCD-K177Q, so that kcat/Km(ATP) is relatively unaffected. The mutant enzyme is impaired in its ability to unwind DNA in an assay where single-stranded DNA is trapped by the single-stranded DNA binding protein and subsequently degraded by S1 nuclease. The mutant enzyme also produces fewer acid-soluble DNA nucleotides per ATP hydrolyzed than does the wild type, at low ATP concentrations (less than 20 microM).