Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group

1. The formation of the non-enzymic adduct of NAD(+) and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD(+). 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD(+) and sulphite was investigated. The u.v. spectrum of the NAD-sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD-sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO(3) (-)=SO(3) (2-)+H(+)), the enzyme-bound adduct showed a bell-shaped pH-stability curve, which indicated that an enzyme side chain of pK=6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated we conclude that the protein group involved is histidine-195. 4. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD(+) and oxalate suggested that an enzyme group is protonated when this complex forms. 5. The rate at which NAD(+) binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD(+) by rapid reaction with sulphite. The rate of NAD(+) dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward V(max.). No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD(+).     

The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide.

1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.     

Site-specific alteration of Gly-24 in streptokinase: its effect on plasminogen activation

Oligonucleotide-directed mutagenesis was carried out to replace glycine-24 of streptokinase with histidine, glutamic acid, or alanine. Substitutions with either histidine or glutamic acid resulted in almost complete loss of streptokinase activity but streptokinase replaced with alanine retained its activity. Although streptokinases with histidine-24 or glutamic acid-24 bound normally to human plasminogen, they were not able to generate active plasmin, whereas those with alanine-24 or glycine-24 (wild-type) could generate active plasmin. The results indicate that the small, uncharged alkyl group side-chain on the 24th amino acid residue of streptokinase is indispensable for the activity of the human plasminogen-streptokinase complex.     

Biosynthesis of bacterial glycogen. Mutagenesis of a catalytic site residue of ADP-glucose pyrophosphorylase from Escherichia coli.

Site-directed mutagenesis was used to explore the role of Lys-195 in ADP-glucose pyrophosphorylase from Escherichia coli. This residue, which is conserved in every bacterial and plant source sequenced to date, was originally identified as a potential catalytic site residue by covalent modification studies. Mutation of Lys-195 to glutamine produces an enzyme whose Km for glucose 1-phosphate is 600-fold greater than that measured for the wild-type enzyme. The effect on glucose 1-phosphate is very specific since kinetic constants measured for ATP, Mg2+, and the allosteric activator, fructose 1,6-bisphosphate, are unchanged relative to those measured for the wild-type enzyme. Furthermore, the catalytic rate constant, Kcat, for the glutamine mutant is similar to that of the wild-type enzyme. Taken together, the results suggest a role for Lys-195 in binding of glucose 1-phosphate and exclude its role as a participant in the rate-determining step(s) in the catalytic reaction mechanism. To further study the effect of charge, shape, size, and hydrophobicity of the amino acid residue at position 195, a series of mutants were prepared including arginine, histidine, isoleucine, and glutamic acid. In every case, the kinetic constants measured for ATP, Mg2+, and fructose 1,6-bisphosphate were similar to wild-type constants, reinforcing the notion that this residue is responsible for a highly localized effect at the glucose 1-phosphate-binding site and also suggesting that the protein can accommodate a wide range of substitutions at this position without losing its global folding properties. Thermal stability measurements corroborate this finding. The mutations did, however, produce a range of glucose 1-phosphate Km values from 100- to 10,000-fold greater than wild-type, which indicate that both size and charge properties of lysine are essential for proper binding of glucose 1-phosphate at the catalytic site. AMP binding was also affected by the nature of the mutation at position 195. A model for glucose 1-phosphate, ATP, and AMP binding is presented.     

Kinetic and spectroscopic studies of Tritrichomonas foetus low-molecular weight phosphotyrosyl phosphatase. Hydrogen bond networks and electrostatic effects

Virtually all of the eukaryotic low-molecular weight protein tyrosine phosphatases (LMW PTPases) studied to date contain a conserved, high-pK(a) histidine residue that is hydrogen bonded to a conserved active site asparagine residue of the phosphate binding loop. However, in the putative enzyme encoded by the genome of the trichomonad parasite Tritrichomonas foetus, this otherwise highly conserved histidine is replaced with a glutamine residue. We have cloned the gene, expressed the enzyme, demonstrated its catalytic activity, and examined the structural and functional roles of the glutamine residue using site-directed mutagenesis, kinetic measurements, and NMR spectroscopy. Titration studies of the two native histidine residues in the T. foetus enzyme as monitored by (1)H NMR revealed that H44 has a pK(a) of 6.4 and H143 has a pK(a) of 5.3. When a histidine residue was introduced in place of the native glutamine at position 67, a pK(a) of 8.2 was measured for this residue. Steady state kinetic methods were employed to study how mutation of the native glutamine to alanine, asparagine, and histidine affected the catalytic activity of the enzyme. Examination of k(cat)/K(m) showed that Q67H exhibits a substrate selectivity comparable to that of the wild-type (WT) enzyme, while Q67N and Q67A show reduced activity. The effect of pH on the reaction rate was examined. Importantly, the pH-rate profile of the WT TPTP enzyme revealed a much more clearly defined acidic limb than that which can be observed for other wild-type LMW PTPases. The pH-rate curve of the Q67H mutant shows a shift to a lower pH optimum relative to that seen for the wild-type enzyme. The Q67N and Q67A mutants showed curves that were shifted to higher pH optima. Although the active site of this enzyme is likely to be similar to that of other LMW PTPases, the hydrogen bonding and electrostatic changes afford new insight into factors affecting the pH dependence and catalysis by this family of enzymes.     

The chemical reactivity of the histidine-195 residue in lactate dehydrogenase thiomethylated at the cysteine-165 residue.

The specific thiomethylation of cysteine-165 (insertion of a methylthio group, CH3-S-) in pig heart lactate dehydrogenase results in a decreased affinity for carbonyl ligands that is accompanied by a decreased nucleophilic reaction of histidine-195 with diethyl pyrocarbonate. The rate constants at 10 degrees C for the modification of native and thiomethylated lactate dehydrogenase by diethyl pyrocarbonate were 173 M-1 . s-1 and 8.7 M-1 . s-1 respectively. It was found that 0.86 +/- 0.07 histidine residue per subunit reacted with diethyl pyrocarbonate in thiomethylated lactate dehydrogenase. This reaction was not affected in the enzyme-NADH binary complex, but was diminished in the enzyme-NADH-oxamate ternary complex. In the enzyme-NADH complex the reaction of diethyl pyrocarbonate was controlled by two groups with pKa 6.8 and 7.9. The decreased reactivity of histidine-195 was selective in thiomethylated lactate dehydrogenase, since the reactivity of arginine and/or lysine residues was enhanced.     

The engineering of a more thermally stable lactate dehydrogenase by reduction of the area of a water-accessible hydrophobic surface

A site-directed mutant of Bacillus stearothermophilus lactate dehydrogenase (lactate:NAD+ oxidoreductase, EC 1.1.1.27) has been engineered in which the conserved hydrophobic residue isoleucine-250 has been replaced by the more hydrophilic residue asparagine. This isoleucine forms a large part of a water-accessible, hydrophobic surface in the active site of the apo-enzyme which is covered by the B-face of the nicotinamide ring when coenzymes are bound. Reduction in the area of this hydrophobic surface results in the mutant tetramer being more thermally stable than the wild-type enzyme.     

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.     

Characterization of human UDP-glucose dehydrogenase reveals critical catalytic roles for lysine 220 and aspartate 280

Human UDP-glucose dehydrogenase (UGDH) is a homohexameric enzyme that catalyzes two successive oxidations of UDP-glucose to yield UDP-glucuronic acid, an essential precursor for matrix polysaccharide and proteoglycan synthesis. We previously used crystal coordinates for Streptococcus pyogenes UGDH to generate a model of the human enzyme active site. In the studies reported here, we have used this model to identify three putative active site residues: lysine 220, aspartate 280, and lysine 339. Each residue was site-specifically mutagenized to evaluate its importance for catalytic activity and maintenance of hexameric quaternary structure. Alteration of lysine 220 to alanine, histidine, or arginine significantly impaired enzyme function. Assaying activity over longer time courses revealed a plateau after reduction of a single equivalent of NAD+ in the alanine and histidine mutants, whereas turnover continued in the arginine mutant. Thus, one role of this lysine may be to stabilize anionic transition states during substrate conversion. Mutation of aspartate 280 to asparagine was also severely detrimental to catalysis. The relative position of this residue within the active site and dependence of function on acidic character point toward a critical role for aspartate 280 in activation of the substrate and the catalytic cysteine. Finally, changing lysine 339 to alanine yielded the wild-type Vmax, but a 165-fold decrease in affinity for UDP-glucose. Interestingly, gel filtration of this substrate-binding mutant also determined it was a dimer, indicating that hexameric quaternary structure is not critical for catalysis. Collectively, this analysis has provided novel insights into the complex catalytic mechanism of UGDH.     

Yeast methionine aminopeptidase I. Alteration of substrate specificity by site-directed mutagenesis

In eukaryotes, two isozymes (I and II) of methionine aminopeptidase (MetAP) catalyze the removal of the initiator methionine if the penultimate residue has a small radius of gyration (glycine, alanine, serine, threonine, proline, valine, and cysteine). Using site-directed mutagenesis, recombinant yeast MetAP I derivatives that are able to cleave N-terminal methionine from substrates that have larger penultimate residues have been expressed. A Met to Ala change at 329 (Met206 in Escherichia coli enzyme) produces an average catalytic efficiency 1.5-fold higher than the native enzyme on normal substrates and cleaves substrates containing penultimate asparagine, glutamine, isoleucine, leucine, methionine, and phenylalanine. Interestingly, the native enzyme also has significant activity with the asparagine peptide not previously identified as a substrate. Mutation of Gln356 (Gln233 in E. coli MetAP) to alanine results in a catalytic efficiency about one-third that of native with normal substrates but which can cleave methionine from substrates with penultimate histidine, asparagine, glutamine, leucine, methionine, phenylalanine, and tryptophan. Mutation of Ser195 to alanine had no effect on substrate specificity. None of the altered enzymes produced cleaved substrates with a fully charged residue (lysine, arginine, aspartic acid, or glutamic acid) or tyrosine in the penultimate position.     

Properties of the histidine residues of indole-chymotrypsin. Implications for the activation process and catalytic mechanism.

The use of a linear free-energy relationship shows that both histidine residues of alpha-chymotrypsin and chymotrypsinogen are super-reactive toward 1-fluoro-2,4-dinitrobenzene. The binding of indole to the specificity site of alpha-chymotrypsin causes both histidine residues to become less reactive. On the basis of these results and those from X-ray-crystallographic studies, the following conclusions are made. (1) The super-reactivity of the catalytic-site histidine-57 is due to charge transfer from aspartic acid-102 by means of hydrogen bonding. (2) The aspartic acid-102-histidine-57-serine-195 'charge-relay' system is not complete in the zymogen or native enzyme and only on binding of a suitable substrate or ligand to the specificity site of the enzyme is the charge transfer to serine-195 completed. (3) The lack of substantial enzymic activity in the zymogen is due to the absence of a completed specificity site, and therefore it cannot bind suitable substrates or ligands to induce completion of the charge-relay system.     

Site-directed mutagenesis of the essential arginine of the formate dehydrogenase active centre

Sequence alignment shows that residue Arg 284 (according to the numbering of the residues in formate dehydrogenase, FDH, from the methylotrophic bacterium Pseudomonas sp. 101) is conserved in NAD-dependent FDHs and D-specific 2-hydroxyacid dehydrogenases. Mutation of Arg 284 to glutamine and alanine results in a change of the catalytic, thermodynamic and spectral properties of FDH. In comparison to wild-type, the affinity of the mutants for the substrate (K(formate)m) or the transition state analogue (K(azide)i) decreases and correlates with the ability of the side chain of residue 284 to form H-bonds. In contrast, the affinity for the coenzyme (K(NAD)d or K(NAD)m) is either not affected or increases and correlates inversely with the partial positive charge of the side chain. The temperature dependence of circular dichroism (CD) spectra of the wild-type FDH and its Ala mutant has been studied over the 5-90 degrees C temperature range. Both proteins reveal regions of enhanced conformational mobility at the predenaturing temperatures (40-55 degrees C) associated with a change of enzyme kinetic parameters and a co-operative transition around 55-70 degrees C which is followed by the loss of enzyme activity. CD spectra of the wild-type and mutant proteins were deconvoluted and contributions from various types of secondary structure estimated. It is shown that the co-operative transition at 55-70 degrees C in the FDH protein globule is triggered by a loss of alpha-helical secondary structure. The results confirm the conclusion, from the crystal structures, that Arg 284 is directly involved in substrate binding. In addition this residue seems to exert a major structural role by supporting the catalytic conformation of the enzyme active centre.     

Histidine-15: an important role in the cytotoxic activity of human tumor necrosis factor

The amino acids that are required for the cytotoxic activity of recombinant human tumor necrosis factor-alpha (TNF) were investigated by chemical modification and oligonucleotide-directed site-specific mutagenesis. TNF contains three histidine residues, located at positions 15, 73 and 78. The histidine-specific reagent diethylpyrocarbonate (DEP) was used to chemically modify TNF. The chemical inactivation of the in vitro cytotoxic activity of this lymphokine (using murine L929 target cells) was found to be time- and dose-dependent. Inactivated TNF failed to compete with fully bioactive [125I]TNF for human MCF-7 target cell receptors. Mutant polypeptides of TNF were genetically engineered by oligonucoleotide-directed site-specific mutagenesis. The cytotoxicity of a double histidine mutant, in which histidine-73 and histidine-78 were replaced with glutamine, was not altered and was chemically inactivated by DEP. Substituting glutamine for histidine-15 resulted in 10-15% of the wild-type bioactivity. Replacing histidine-15 with either asparagine, lysine or glycine resulted in a biologically inactive molecule. The data show that the histidine residue at position 15 is an amino acid that is required for the cytotoxic activity of TNF.     

An evolutionarily conserved alternate metal ligand is important for activity in α-isopropylmalate synthase from Mycobacterium tuberculosis

Members of the DRE-TIM metallolyase superfamily rely on an active-site divalent cation to catalyze various reactions involving the making and breaking of carbon–carbon bonds. While the identity of the metal varies, the binding site is well-conserved at the superfamily level with an aspartic acid and two histidine residues acting as ligands to the metal. Previous structural and bioinformatics results indicate that the metal can adopt an alternate architecture through the addition of an asparagine residue as a fourth ligand. This asparagine residue is strictly conserved in all members of the DRE-TIM metallolyase superfamily except fungal homocitrate synthase (HCS-lys) where it is replaced with isoleucine. The role of this additional metal ligand in α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been investigated using site-directed mutagenesis. Substitution of the asparagine ligand with alanine or isoleucine results in inactive enzymes with respect to α-isopropylmalate formation. Control experiments suggest that the substitutions have not drastically affected the enzyme's structure indicating that the asparagine residue is essential for catalysis. Interestingly, all enzyme variants retained acetyl CoA hydrolysis activity in the absence of α-ketoisovalerate, similar to the wild-type enzyme. In contrast to the requirement of magnesium for α-isopropylmalate formation, hydrolytic activity could be inhibited by the addition of magnesium chloride in wild-type, D81E, and N321A MtIPMS, but not in the other variants studied. Attempts to rescue loss of activity in N321I MtIPMS by mimicking the fungal HCS active site through the D81E/N321I double variant were unsuccessful. This suggests epistatic constraints in evolution of function in IPMS and HCS-lys enzymes.     

Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test.

The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.     

(S)-Mandelate dehydrogenase from Pseudomonas putida: mutations of the catalytic base histidine-274 and chemical rescue of activity.

(S)-Mandelate dehydrogenase from Pseudomonas putida, an FMN-dependent alpha-hydroxy acid dehydrogenase, oxidizes (S)-mandelate to benzoylformate. The generally accepted catalytic mechanism for this enzyme involves the formation of a carbanion intermediate. Histidine-274 has been proposed to be the active-site base that abstracts the substrate alpha-proton to generate the carbanion. Histidine-274 was altered to glycine, alanine, and asparagine. All three mutants were completely inactive. The mutants were able to form adducts with sulfite, though with much weaker affinity than the wild-type enzyme. Binding of the inhibitor, (R)-mandelate, was not greatly affected by the mutation, unlike that of the substrate, (S)-mandelate, indicating that H274 plays a role in substrate binding. The activity of H274G and, to a lesser extent, H274A could be partially restored by the addition of exogenous imidazoles. The maximum rescued activity for H274G with imidazole was approximately 0.1% of the wild-type value. Saturation kinetics obtained for rescued activity suggest that formation of a ternary complex of imidazole, enzyme, and substrate is required for catalysis. pH-dependence studies confirm that the free base form of imidazole is the rescue agent. An earlier study of pH profiles of the wild-type enzyme indicated that deprotonation of a residue with a pK(a) of 5.5 in the free enzyme was essential for activity (Lehoux, I. E., and Mitra, B. (1999) Biochemistry 38, 5836-5848). Data obtained in this work confirm that the pK(a) of 5.5 belongs to histidine-274.     

Kinetic characterization and ligand binding studies of His351 mutants of Bacillus anthracis adenylate cyclase

Edema factor is a calmodulin dependent adenylyl cyclase secreted as one of the primary exotoxins by Bacillus anthracis. A histidine residue at position 351 located in its active site has been implicated in catalysis but direct evidence of its functional role is still lacking. In the present study, we introduced mutations in full-length edema factor (EF) to generate alanine (H351A), asparagine (H351N), and phenylalanine (H351F) variants. Spectral analysis of these variants displayed no gross structural deformities. Kinetic characterization showed that the adenylyl cyclase activity of H351N and H351F mutants decreased 34- and 40-fold, respectively, whereas H351A mutant completely lost activity. K(m) and K(i) values for ATP, pH activity profiles, and calmodulin activation curves of asparagine and phenylalanine mutants were not altered markedly. This kinetic data corroborated our ligand binding studies. Apparent K(d) values for calmodulin and ATP binding were found to be similar for wild-type EF and these active site variants. The effective substitution of H351 by asparagine and phenylalanine, albeit at a greatly reduced K(cat), without perturbing the ATP binding highlights the importance of this residue in transition-state stabilization. This was also evident from the positive free energy difference calculated for these mutants. However, equilibrium dialysis experiments revealed noticeable increase in ATP binding constant of H351A mutant, suggesting an additional role of H351 in precise substrate binding in the catalytic pocket. This is the first comprehensive study that describes the kinetic and ligand binding properties of H351 mutants and validates the importance of this residue in EF catalysis.     

The use of ternary complexes to study ionizations and isomerizations during catalysis by lactate dehydrogenase

1. The binding of oxamate to pig heart and pig muscle isoenzymes of lactate dehydrogenase in the presence of NADH was studied by fluorescence titration. The dissociation constant of oxamate from the heart enzyme complex is 3mum and from the muscle isoenzyme 25mum at pH5. These values quantitatively increase with pH as predicted if oxamate can bind only to the enzyme-NADH complex if a group with pK6.9 is protonated. There are four non-interacting oxamate-binding sites per tetramer. 2. o-Nitrophenylpyruvate is a poor substrate for both isoenzymes but has a reasonable affinity to the heart isoenzyme. Initially, it forms an enzyme-NADH-substrate complex, which can be detected either by protein-fluorescence quenching or by NADH-fluorescence quenching. The pH-dependence of the dissociation constant of nitrophenylpyruvate also shows that this ternary complex can only form if a group with pK6.8 is protonated. Taken with the results of chemical-modification experiments, these results allow the pK of 6.8 to be assigned to a system probably involving the imidazole side chain of histidine-195. Formation of a ternary complex from a binary one at pH8 is predicted to result in a proton being taken up from solution. 3. Isotope-effect studies with NADH and its deuterium analogue show that the rapidly formed ternary complex with o-nitrophenylpyruvate slowly isomerizes to give an active ternary complex, which then rapidly decomposes to NAD(+). The isomerization is pH-independent, and it is suggested that histidine-195 is still protonated in the activated ternary complex, which is present before hydride transfer. 4. All four subunits of the enzyme are kinetically equivalent with respect to the oxidation of bound NADH by o-nitrophenylpyruvate. 5. A partial mechanism for the enzyme is described which emphasizes the isomerizations and ionizations involved in forming the reduced ternary complex at pH6 and 8.     

Electrophilic catalysis in triosephosphate isomerase: the role of histidine-95

Electrophilic catalysis by histidine-95 in triosephosphate isomerase has been probed by using Fourier transform infrared spectroscopy and X-ray crystallography. The carbonyl stretching frequency of dihydroxyacetone phosphate bound to the wild-type enzyme is known to be 19 cm-1 lower (at 1713 cm-1) than that of dihydroxyacetone phosphate free in solution (at 1732 cm-1), and this decrease in stretching frequency has been ascribed to an enzymic electrophile that polarizes the substrate carbonyl group toward the transition state for the enolization. Infrared spectra of substrate bound to two site-directed mutants of yeast triosephosphate isomerase in which histidine-95 has been changed to glutamine or to asparagine show unperturbed carbonyl stretching frequencies between 1732 and 1742 cm-1. The lack of carbonyl polarization when histidine-95 is removed suggests that histidine-95 is indeed the catalytic electrophile, at least for dihydroxyacetone phosphate. Kinetic studies of the glutamine mutant (H95Q) have shown that the enzyme follows a subtly different mechanism of proton transfers involving only a single acid-base catalytic group. These findings suggest an additional role for histidine-95 as a general acid-base catalyst in the wild-type enzyme. The X-ray crystal structure of the H95Q mutant with an intermediate analogue, phosphoglycolohydroxamate, bound at the active site has been solved to 2.8-A resolution, and this structure clearly implicates glutamate-165, the catalytic base in the wild-type isomerase, as the sole acid-base catalyst for the mutant enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)