Assignment of the histidine proton magnetic resonance peaks of soybean trypsin inhibitor (Kunitz) by a differertial deuterium exchange technique.

Deuterium exchange at the C(2)-H position of the two histidine residues of native soybean trypsin inhibitor (Kunitz) in 2-H2O was followed by 1-H nuclear magnetic resonance (NMR) spectroscopy. The two histidine residues of soybean trypsin inhibitor exchange at significantly different rates at pH* 5.00, 40 degrees. Half-times observed were: peak H1, t1/2=61 plus or minus 2 days; peak H2, T1/2=24 plus or minus 2 days. Differentially deuterated soybean trypsin inhibitor was cleaved by cyanogen bromide into two fragments each containing one histidine residue. The deuterium content of the histidine residue of each separated fragment was analyzed by 1H NMR spectroscopy. Hisidine-71 in fragment 1-114 showed approximately twice the deuterium content of His-157 in fragment 115-181. These results lead to the assignment of 1H NMR peak H1 to His-157 and peak H2 to His-71. These assignments were extended to the histidine peaks of trypsin-modified soybean trypsin inhibitor by converting the differentially deuterated virgin soybean trypsin inhibitor to the modified form. The correlation of histidine peaks in virgin amd modified soybean trypsin inhibitors was the same as proposed earlier on the basis of pK arguments. The results demonstrate that His-71 is the residue whose pK value is raised from 5.27 to 5.91 on trypsin modification of soybean trypsin inhibitor [Markley, J. L., (1973), Biochemistry 12, 2245].     

Enzymic and physicochemical properties of Streptomyces griseus trypsin.

Streptomyces griseus trypsin has been isolated from Pronase by ion-exchange chromatography on CM-Sephadex and SE-Sephadex. The isolated enzyme was homogeneous by the criteria tested except for a low degree of contamination by an enzyme with nontryptic activity. The latter could be partially resolved by chromatography on Bio-Rex 70. The molar absorbancy at 280 nm was found to be 3.96 times 10-4 M-1/cm and the E1cm1% was found to be 17.3. The molecular weight was 22,800 plus or minus 800. The enzyme was found to be stable at 0 degrees from pH 2 to 10. At 30 degrees the enzyme was maximally stable at pH 3-4 and significantly stabilized in the neutral and alkaline range by 15 mM Ca2+. Some evidence was obtained for a reversible denaturation of the enzyme at pH 12.0 and 2.0. The K-m for N-alpha-benzoyl-L-arginine ethyl ester at pH 8.0 in 20 mM CaCl2-0.1 M KCl-10 mM Tris-HCl buffer at 30 degrees was found to be 7.7 plus or minus 1.9 times 10-6 M and the esterase activity was observed to be dependent on an ionizing group with pK-a equals 5.85. In 2H2O this pKa was increased to 6.35 and the rate of hydrolysis dicreased threefold. The rate of hydrolysis was independent of pH between 8 and 10. The inhibition of the enzyme with L-1-chloro-3-tosylamido-4-phenyl-2-butanone was shown to be associated with the alkylation of its single histidine residue. This residue is present in a homologous amino acid sequence as the active-site histidine in trypsin and chymotrypsin. Optical rotatory dispersion and circular dichroism measurements over the pH range 5.3-10.5 indicated no significant conformational change until the pH was increased above 10.1. The observation that, under the conditions tested, acetylation and carbamylation of the NH2-terminal valine were incomplete is consistent with the view that this group is buried as an ion pair and only becomes available for deprotonation and reaction upon denaturation of the enzyme at pH values greater than 10.0.     

Chemical and kinetic evidence for an essential histidine in the phosphotriesterase from Pseudomonas diminuta

The pH rate profile for the hydrolysis of diethyl-p-nitrophenyl phosphate catalyzed by the phosphotriesterase from Pseudomonas diminuta shows a requirement for the deprotonation of an ionizable group for full catalytic activity. This functional group has an apparent pKa of 6.1 +/- 0.1 at 25 degrees C, delta Hion of 7.9 kcal/mol, and delta Sion of -1.4 cal/K.mol. The enzyme is not inactivated in the presence of the chemical modification reagents dithiobis-(2-nitrobenzoate), methyl methane thiosulfonate, carbodiimide, pyridoxal, butanedione, or iodoacetic acid and thus cysteine, asparate, glutamate, lysine, and arginine do not appear to be critical for catalytic activity. However, the phosphotriesterase is inactivated completely with methylene blue, Rose Bengal, or diethyl pyrocarbonate. The enzyme is not inactivated by diethyl pyrocarbonate in the presence of bound substrate analogs, and inactivation with diethyl pyrocarbonate is reversible upon addition of neutralized hydroxylamine. The modification of a single histidine residue by diethyl pyrocarbonate, as shown by spectrophotometric analysis, is responsible for the loss of catalytic activity. The pKinact for diethyl pyrocarbonate modification is 6.1 +/- 0.1 at 25 degrees C. These results have been interpreted to suggest that a histidine residue at the active site of phosphotriesterase is facilitating the reaction by general base catalysis.     

Thermostable D-amino acid aminotransferase from a thermophilic Bacillus species. Purification, characterization, and active site sequence determination

D-Amino acid aminotransferase was found in several thermophilic Bacillus species and purified to homogeneity from the best producer, Bacillus sp. YM-1, which was newly isolated from a sauna dust. The enzyme has a molecular weight of about 62,000 and consists of two subunits identical in molecular weight (30,000). It catalyzes transamination between various D-amino acids and alpha-keto acids, although the substrate specificity is narrower than the enzyme from the mesophile, Bacillus sphaericus (Yonaha, K., Misono, H., Yamamoto, T., and Soda, K. (1975) J. Biol. Chem. 250, 6983-6989). The Bacillus sp. YM-1 enzyme is most active at 60 degrees C and stable at high temperatures. Automated Edman degradation provided the N-terminal sequence of the first 20 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 3 amino acids. The amino acid sequence in the vicinity of the lysyl residue, Lys(Pxy), that binds pyridoxal 5'-phosphate was determined as Cys-Asp-Ile-Lys(Pxy)-Ser-Leu-Asn-Leu-Leu-Gly-Ala-Val-Leu-Ala-Lys- from the pyridoxyl peptide obtained by digestion with trypsin. The active site sequence is markedly different from those of L-amino acid aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes.     

The pH dependence of the hydrolysis of benzoyl-L-arginine ethyl ester in cooled mixed solvents.

Tables of protonic activity (paH) of a number of buffers, determined in mixed solvents and at subzero temperatures, are reported for the following media: water-1,2-propanediol, water-glycerol, and water-dimethylsulfoxide (50:50, in volume). These data with those previously reported allowed us to study enzymic reactions under these conditions. The paH dependence of the tryptic hydrolysis of benzoyl-L-arginine ethyl ester has been studied in the presence of organic solvents (methanol, ethylene glycol, 1,2-propanediol, glycerol, and dimethylsulfoxide, all 50% by volume) between 20 and -20 degrees. The results have allowed us to show the validity of our paH scales in mixed solvents. The paH profiles obtained under these conditions are similar to those observed in pure water at 20 degrees. They are shifted nevertheless by both solvent and temperature. Such shifts are interpreted in terms of the effects of solvents and temperature on pKES on the basis of the conclusions drawn from a study of the effect of these variables on small dissociable molecules. The results obtained under these conditions of solvents and temperature are consistent with the presence at the active site of the enzyme of a histidine residue, and thus provide, concerning the solvent effect, a direct verification of the method of Findlay et al. (Findlay, D., Mathias, A. P., and Rabin, B. R. (1962) Biochem. J. 85, 139-144). On the other hand, the large temperature interval provided by the low temperature procedure, allows us to vary significantly the pK of ionizable groups of the enzymes and thus makes possible their identification, on the basis of their enthalpy of ionization.     

Equilibrium of Bowman-Birk inhibitor association with trypsin and alpha-chymotrypsin.

Association constants, enthalpies, and stoichiometries of Bowman-Birk soybean inhibitor for trypsin and alpha-chymotrypsin were measured in the pH range 4-8 at 25 degrees, 0.01 M Ca2+. The results are quoted in terms of moles of protease active sites, from active site titration. Enthalpies were obtained from calorimetry. The inhibitor was modified by carboxyl group modification, and by tryptic and chymotryptic attack. Association thermodynamics and stoichiometries of the modified inhibitors with both proteases were also determined. There is one independent site for each protease on the inhibitor protein. Modification decreases association to some extent, but does not appear to change stoichiometry or protease binding site independency. In the pH 4 region the association enthalpies are endothermic, of the order 6 kcal/mol for both trypsin and chymotrypsin. With increasing pH, the enthalpies decrease and become exothermic at pH 8 for chymotrypsin. Positive entropies, 50 cal mol-1 deg-1, occur at pH 4-5. They decrease as pH increases, but are always positive in sign. The observed to accompany the overall reaction, such as H+ transfer steps. The enthalpies and entropies probably compensate over the pH range 4-8, with a characteristic temperature of 390 plus or minus 30 degrees K. Estimates were made of the macromolecular Coulomb charge products in inhibitor-protease interaction. These range from about +5 to -60, over pH range 4-8, depending on the protease. Although intermolecular Coulombic forces cannot be easily delineated at the specific side chain level, they may operate at the macromolecule level.     

Gene identification and characterization of the pyridoxine degradative enzyme 4-pyridoxic acid dehydrogenase from the nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099

The gene encoding 4-pyridoxic acid dehydrogenase was identified as mlr6792 in a chromosome of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099. The enzyme is the fourth enzyme in the vitamin B(6) (pyridoxine)-degradation pathway I. The recombinant enzyme with a his-tag over-expressed in Escherichia coli cells was a membrane-bound protein, and purified to homogeneity. The enzyme was a monomeric protein with a molecular weight of 59,000, and a flavoprotein containing one mole of FAD per mole of subunit. The optimum pH and temperature, and K(m) for 4-pyridoxic acid were pH 8.5 and 30 degrees C, and 29 muM, respectively. The enzyme was a glucose-methanol-choline (GMC) family protein with two signature patterns, FAD-binding residues, a putative active site histidine residue and a probable transmembrane segment.     

Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis

Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae) were purified on an aprotinin-agarose column equilibrated with 0.01 M Tris-HCl containing 5 mM CaCl2 (pH 7.5). The yield was 66.7% with a purification factor of 107 and a final specific activity of 6.88 mM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). The purified fraction showed three bands with proteolytic activity and molecular weights of 66,000, 71,000 and 91,000 (sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)). Enzyme specificity assays were carried out using seven synthetic peptides containing 13 amino acid residues, but differing only on the 5th residue (K, R, Y, L, W or P). Peptide cleavage takes place only with amino acids K or R at the 5th position, which is typical of trypsin. The partially purified enzymes hydrolyzed casein and the synthetic trypsin substrates L-BApNA and N-alpha-p-tosyl-L-Arg methyl ester (L-TAME). Higher activity was observed at pH 8.5 and 35 degrees C when using L-BApNA as substrate and at pH 8.0 and 30 degrees C when using L-TAME. Maximum enzyme activity against L-BApNA was obtained with 20 mM CaCl2 in the reaction mixture. The partially purified enzymes showing trypsin activity were sensitive to inhibition by ethylenediaminetetraacetic acid (EDTA), phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. Highest inhibition was obtained with TLCK and benzamidine. KM values obtained were 0.32 mM for L-BApNA and 52.5 microM for L-TAME.     

Assessing the roles of essential functional groups in the mechanism of homoserine succinyltransferase

Homoserine acyltransferases catalyze the commitment step to methionine and other important biological precursors which make this class of enzymes essential for the survival of bacteria, plants and fungi. This class of enzymes is not found in humans, making them an attractive new target for antimicrobial design. Homoserine O-succinyltransferase (HST) is a representative from this class, with little known about the key amino acids involved in substrate specificity and catalysis. HST from Escherichia coli has been cloned, purified and kinetically characterized. Through site-directed mutagenesis and steady-state kinetic studies the residues that comprise a catalytic triad for HST, the catalytic cysteine nucleophile, an active site acid-base histidine, and the base orienting glutamate, have been identified and characterized. Several residues which confer substrate specificity for both homoserine and succinyl-CoA have also been identified and kinetically evaluated. Mutations of an active site glutamate to either aspartate or alanine drastically increase the K(m) for homoserine, assigning this glutamate to a binding role for the alpha-amino group of homoserine. An active site arginine orients the carboxyl moiety of homoserine, while the carboxyl moiety of succinyl-CoA is positioned for catalysis by a lysine residue. Removing functionality at either of these positions alters the enzyme's ability to effectively utilize homoserine or succinyl-CoA, respectively, reflected in an increased K(m) and decreased catalytic efficiency. The data presented here provides new details of the catalytic mechanism of succinyltransferases, resolves a controversy between alternative mechanistic hypotheses, and provides a starting point for the development of selective inhibitors of HST.     

A structural and mechanistic comparison of pyridoxal 5'-phosphate dependent decarboxylase and transaminase enzymes.

Stereochemical studies of three pyridoxal phosphate dependent decarboxylases and serine hydroxymethyltransferase have allowed the dispositions of conjugate acids that operate at the C alpha and C-4' positions of intermediate quinoids to be determined. Kinetic work with the decarboxylase group has determined that two different acids are involved, a monoprotic acid and a polyprotic acid. The use of solvent kinetic isotope effects allowed the resolution of chemical steps in the reaction coordinate profile for decarboxylation and abortive transamination and pH-sensitivities gave the molecular pKa of the monoprotic base. Thus the epsilon-ammonium group of the internal aldimine-forming lysine residue operates at C-4'-si-face of the coenzyme and the imidazolium side chain of an active site histidine residue protonates at C alpha from the 4'-si-face. Histidine serves two other functions, as a base in generating nitrogen nucleophiles during both transaldimination processes and as a binding group for the alpha-carboxyl group of substrates. The latter role for histidine was determined by comparison of the sequences for decarboxylase active site tetrapeptides (e.g. -S-X-H-K-) with that for aspartate aminotransferase (e.g. -S-X-A-K-) where it was known, from X-ray studies, that the serine and lysine residues interact with the coenzyme. By using the Dunathan Postulate, the conformation of the external aldimine was modified, and without changing the tetrapeptide conformation, the alanine residue was altered to a histidine. This model for the active site of a pyridoxal dependent decarboxylase was consistent with all available stereochemical and mechanistic data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of association of a specific aldehyde inhibitor, leupeptin, with bovine trypsin

Leupeptin (acyl peptidyl-L-argininal) is a potent inhibitor of trypsin and related proteases. We analyzed the association of leupeptim with bovine trypsin kinetically, assuming that it proceeds by a pathway which involves two steps: E + I in equilibrium K1 Complex I k-2 in equilibrium k+2 Complex II. The observed dissociation constant (K1) for the first step was 1.24 X 10(-3) M (at pH 8.2 15 degrees C) and the two first-order rate constants (k+2 and k-2) were 166 s-1 and 1.75 X 10(-3.s-1, respectively (at pH 8.2, 15 degrees C). The dissociation constant (Kd) for the whole process was calculated from these parameters to be 1.34 X 10(-8) M. This value is compatible with that determined directly by an independent static method (2.36 X 10(-8) M). We also measured Kd for the leupeptine complex of anhydrotrypsin, a trypsin derivative in which the active-site hydroxyl group is missing. The observed value was about 5 orders of magnitude larger than Kd and was rather similar to K1 in native trypsin. A elupeptin isomer which contains a D-argininal residue did not show strong affinity towards trypsin. These findings suggest that complex II consists of a covalent hemiacetal adduct formed between the serine hydroxyl group in the enzyme active site and the aldehyde group in the inhibitor. The pH dependencies of the dissociation constant and other parameters show that deprotonation of the charge-relay sustem in the active site is important for the formation and stabilization of complex II.     

Evidence for a functional role for histidine in lysyl oxidase catalysis

The pH-dependent kinetics of lysyl oxidase catalysis was examined for evidence of an ionizable enzyme residue which might function as a general base catalyzing proton abstraction previously shown to be a component of the mechanism of substrate processing by this enzyme. Plots of log Vmax/Km for the oxidation of n-hexylamine versus pH yielded pKa values of 7.0 +/- 0.1 and 10.4 +/- 0.1. The higher pKa varied with different substrates, reflecting ionization of the substrate amino group. A van't Hoff plot of the temperature dependence of the lower pKa yielded a value of 6.1 kcal mol-1 for the enthalpy of ionization. This value as well as the pKa of 7.0 are consistent with those of histidine residues previously implicated as general base catalysts in enzymes. Incubation of lysyl oxidase with low concentrations of diethyl pyrocarbonate, a histidine-selective reagent, at 22 degrees C and pH 7.0 irreversibly inhibited enzyme activity by a pseudo first-order kinetic process. The inactivation of lysyl oxidase correlated with spectral and pH-dependent kinetic evidence for the chemical modification of 1 histidine residue/mol of enzyme, the pKa of which was 6.9 +/- 0.1, within experimental error of that seen in the plot of log Vmax/Km versus pH. Enzyme activity was restored by incubation of the modified enzyme with hydroxylamine, consistent with the ability of this nucleophile to displace the carbethoxy group from N-carbethoxyhistidine. The presence of the n-hexylamine substrate largely protected against enzyme inactivation by diethyl pyrocarbonate. These results thus indicate a functional role for histidine in lysyl oxidase catalysis consistent with that of a general base in proton abstraction.     

Evidence for the presence of a critical histidine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.     

Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae

Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.     

Nuclear magnetic resonance titration curves of histidine ring protons. Conformational transition affecting three of the histidine residues of ribonuclease.

NMR titration curves are reported for the 4 histidine residues of ribonuclease A in sodium acetate and for ribonuclease S in sodium acetate, phosphate, and sulfate solutions. Evidence is presented that the imidazole side chain of histidine residue 48 undergoes a conformational change, probably also involving the carboxyl side chain of aspartic acid residue 14. This group is considered to be responsible for the low pH inflection with pKa 4.2 present in the NMR titration curve of the C-2 proton resonance of histidine 48. The NMR titration curves of the active site histidine residues 12 and 119 also exhibit inflections at low pH values, although there is no carboxyl group within 9 A of the imidazole side chain of histidine residue 12 in the structure of ribonuclease S determined by x-ray crystallography (Wyckoff, H. W., Tsernoglou, D., Hanson, A. W. Knox, J. R., Lee, B., and Richards, F. M. (1970) J. Biol. Chem. 245, 305-328). Curve fitting was carried out on 11 sets of NMR titration data using a model in which the 3 histidine residues 12, 119, and 48 are assumed to be affected by a common carboxyl group. The results obtained indicate that such a model with fewer parameters gives as good a representation of the data as the model in which each histidine residue is assumed to interact separately with a different carboxyl group. Therefore, it is concluded that the ionization of aspartic acid residue 14 is indirectly experienced by the active site histidine residues through the conformational change at histidine 48. A model assuming mutual interaction of the active site histidine residues does not account for the low pH inflections in these curves.     

Purification, characterization and reaction mechanism of novel arylsulfotransferase obtained from an anaerobic bacterium of human intestine

A novel type of arylsulfotransferase was purified from Eubacterium A-44, one of the predominant bacteria of human intestine. The enzyme (Mr 315 000) was composed of four identical subunits (Mr 80 000) whose N-terminal amino acids were arginine. pI and optimal pH of the enzyme were 3.9 and 8-9, respectively. The apparent Km for p-nitrophenylsulfate using tyramine as an acceptor substrate and that for tyramine using p-nitrophenylsulfate as a donor substrate were determined to be 0.104 mM and 3.5 mM, respectively. The reaction mechanism of the enzyme was proposed as follows: a donor substrate, p-nitrophenyl [35S]sulfate, combines a histidine residue of the enzyme active site with concomitant release of a phenolic compound, p-nitrophenol. The sulfate group of the histidine residue transfers to a tyrosine group, and then to an acceptor with the binding of another donor to the histidine residue.     

Screening of the active site from water by the incoming ligand triggers catalysis and inhibition in serine proteases

The pKa of the catalytic His57 N(epsilon)H in the tetrahedral complex (TC) of chymotrypsin with trifluoromethyl ketone inhibitors is 4-5 units higher relative to the free enzyme (FE). Such stable TC's, formed with transition state (TS) analog inhibitors, are topologically similar to the catalytic TS. Thus, analysis of this pKa shift may shed light on the role of water solvation in the general base catalysis by histidine. We applied our QM/SCRF(VS) approach to study this shift. The method enables explicit quantum mechanical DFT calculations of large molecular clusters that simulate chemical reactions at the active site (AS) of water solvated enzymes. We derived an analytical expression for the pKa dependence on the degree of water exposure of the ionizable group, and on the total charge in the enzyme AS, Q(A) and Q(B), when the target ionizable functional group (His57 in this study) is in the acidic (A) and basic (B) forms, respectively. Q2(B) > Q2(A) both in the FE and in the TC of chymotrypsin. Therefore, water solvation decreases the relative stability of the protonated histidine in both. Ligand binding reduces the degree of water solvation of the imidazole ring, and consequently elevates the histidine pKa. Thus, the binding of the ligand plays a triggering role that switches on the cascade of catalytic reactions in serine proteases.     

A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.     

Identification of histidine residues at the active site of Megalobatrachus japonicus alkaline phosphatase by chemical modification

Alkaline phosphatase from Megalobatrachus japonicus was inactivated by diethyl pyrocarbonate (DEP). The inactivation followed pseudo-first-order kinetics with a second-order rate constant of 176 M(-1) x min(-1) at pH 6.2 and 25 degrees C. The loss of enzyme activity was accompanied with an increase in absorbance at 242 nm and the inactivated enzyme was re-activated by hydroxylamine, indicating the modification of histidine residues. This conclusion was also confirmed by the pH profiles of inactivation, which showed the involvement of a residue with pK(a) of 6.6. The presence of glycerol 3-phosphate, AMP and phosphate protected the enzyme against inactivation. The results revealed that the histidine residues modified by DEP were located at the active site. Spectrophotometric quantification of modified residues showed that modification of two histidine residues per active site led to complete inactivation, but kinetic stoichiometry indicated that one molecule of modifier reacted with one active site during inactivation, probably suggesting that two essential histidine residues per active site are necessary for complete activity whereas modification of a single histidine residue per active site is enough to result in inactivation.     

A study of ligand binding to spleen myeloperoxidase

The ligand binding properties of spleen myeloperoxidase, a peroxidase formerly called "the spleen green hemeprotein", were studied as functions of temperature and pH, using chloride and cyanide as exogenous ligands. Ligand binding is influenced by a proton dissociable group with a pKa of 4. The protonated, uncharged form of cyanide binds to the unprotonated form of the enzyme, while chloride ion binds to the enzyme when this group is protonated. In both cyanide and chloride binding, the pH-dependent change in the apparent ligand affinity is due to a change in the apparent association rate with pH. The proton dissociable group on the enzyme involved in ligand binding has a delta H value of about 8 kcal . mol-1. The present results suggest that this ionizable group is the imidazole group of a histidine residue located near the ligand binding site.