A survey of the kinetic parameters of class C beta-lactamases. Penicillins.

The interaction between six class C beta-lactamases and various penicillins has been studied. All the enzymes behaved in a very uniform manner. Benzylpenicillin exhibited relatively low kcat. values (14-75 s-1) but low values of Km resulted in high catalytic efficiencies [kcat./Km = 10 X 10(6)-75 X 10(6) M-1.s-1]. The kcat. values for ampicillin were 10-100-fold lower. Carbenicillin, oxacillin cloxacillin and methicillin were very poor substrates, exhibiting kcat. values between 1 x 10(-3) and 0.1 s-1. The Km values were correspondingly small. It could safely be hypothesized that, with all the tested substrates, deacylation was rate-limiting, resulting in acyl-enzyme accumulation.     

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.     

Preparation of chromophoric substrates for the glutamoyl specific staphylococcal protease.

The synthesis of chromophoric substrates allowing an accurate determination of the staphylococcal protease activity is described. BOC-L-Glu-OPh, BOC-L-Phe-L-Glu-OPh, BOC-L-Ala-L-Glu-OPh, BOC-L-Ser-L-Glu-OPh and Z-L-Glu-pNA were prepared. Kinetic parameters of the staphyloccal protease-catalysed hydrolyses of these substrates are compared. In every case the dipeptide ester substrates lead to a lower catalytic efficiency (kcat/Km ratio), compared with either BOC-L-Glu-OPh or Z-L-Glu-OPh, mainly because of an increase in the Km value. Like other serine proteinases, the staphylococcal protease exhibits a high ratio of eeterase to peptidase activity, the kcat/Km ratio being 2.6 X 10(5)-fold higher with the Z-L-Glu-OPh than with the Z-L-Glu-pNA.     

Studies on the activity and activation of rat liver microsomal glutathione transferase, in particular with a substrate analogue series

A number of potential substrates for the microsomal glutathione transferase have been investigated. Out of 11 epoxides tested, only two, i.e. androstenoxide and benzo(a)pyrene-4,5-oxide, were found to be substrates. Upon treatment of the enzyme with N-ethylmaleimide, its activity toward only certain substrates is increased. It appeared upon inspection of the bimolecular rate constants from the corresponding nonenzymatic reactions that the substrates for which the activity is increased are the more reactive ones. This hypothesis was investigated further using a series of para-substituted 1-chloro-2-nitrobenzene derivatives as substrates. Activation was seen only with the more reactive nitro-, aldehyde-, and acetaldehyde-substituted compounds and not with the amide and chloroanalogues, thus demonstrating the predicted effect with a related series of compounds. Interestingly, kcat values are increased 7-20-fold by N-ethylmaleimide treatment, whereas the corresponding kcat/Km value is increased only for the p-nitro derivative. Effective molarity and rate enhancement values were found to increase with decreasing reactivity of the substrate, attaining maximal values of 10(5) M and 10(8), respectively. It is concluded that the glutathione transferases are quite effective catalysts with their less reactive substrates. Hammett rho values for the kcat values of unactivated and activated enzyme were 0.49 and 2.0, respectively. The latter value is close to those found for cytosolic glutathione transferases, indicating that activation changes the catalytic mechanism so that it more closely resembles that of the soluble enzymes. The rho values for kcat/Km values were 3 and 3.5 for the unactivated and activated enzyme, respectively, values close to those observed for the nonenzymatic bimolecular rate constants and thereby demonstrating that these reactions have similar properties. The high coefficients of correlation between resonance sigma- values and all of these parameters demonstrate a strong dependence on substrate electrophilicity, as expected for nucleophilic aromatic substitution.     

Chemical synthesis and papain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide.

The chemical synthesis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide (Z-Lys-pNA) is described in detail. The pH-dependence of the catalytic parameters kcat,' Km and kcat./Km for the papain-catalysed hydrolysis of Z-Lys-pNA are determined. kcat. and Km are pH-independent between pH 5 and pH 7.42, but the pH-dependence of kcat./Km is bell-shaped, decreasing at high and low pH values with pKa values of 7.97 and 4.40 respectively. The catalytic parameters and their pH-dependence are shown to be similar to those reported for other anilide substrates and it is concluded that the Km value of 0.01 mM previously reported [Angelides & Fink (1979) Biochemistry 18, 2355-2369] is incorrect. The possibility of accumulating a tetrahedral intermediate during the papain-catalysed hydrolysis of Z-Lys-pNA is discussed.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Arg292----Val] or [Arg292----Leu] mutation enhances the reactivity of Escherichia coli aspartate aminotransferase with aromatic amino acids

Arg292 of E. coli aspartate aminotransferase was substituted with valine or leucine by site-directed mutagenesis. In comparison with the wild-type enzyme, either of the mutant enzymes showed a decrease by over 5 orders of magnitude of kcat/km values for aspartate and glutamate. This supports the contention that Arg292 is important for determining the specificity of this enzyme for dicarboxylic substrates. In contrast, mutant enzymes displayed a 5- to 10-fold increase in kcat/Km values for aromatic amino acids as substrates. Thus, introduction of an uncharged, hydrophobic side chain into position 292 leads to a striking alteration in substrate specificity of this enzyme, thereby improving catalytic efficiency toward aromatic amino acids.     

Significance of highly conserved aromatic residues in Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase

Undecaprenyl diphosphate synthase catalyzes the sequential condensation of eight molecules of isopentenyl diphosphate (IPP) in the cis-configuration into farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of the bacterial cell wall. This cis-type prenyltransferase exhibits a quite different mode of binding of homoallylic substrate IPP from that of trans-type prenyltransferase [Kharel Y. et al. (2001) J. Biol. Chem. 276, 28459-28464]. In order to know the IPP binding mode in more detail, we selected six highly conserved residues in Regions III, IV, and V among nine conserved aromatic residues in Micrococcus luteus B-P 26 UPP synthase for substitution by site-directed mutagenesis. The mutant enzymes were expressed and purified to homogeneity, and then their effects on substrate binding and the catalytic function were examined. All of the mutant enzymes showed moderately similar far-UV CD spectra to that of the wild-type, indicating that none of the replacement of conserved aromatic residues affected the secondary structure of the enzyme. Kinetic analysis showed that the replacement of Tyr-71 with Ser in Region III, Tyr-148 with Phe in Region IV, and Trp-210 with Ala in Region V brought about 10-1,600-fold decreases in the kcat/Km values compared to that of the wild-type but the Km values for both substrates IPP and FPP resulted in only moderate changes. Substitution of Phe-207 with Ser in Region V resulted in a 13-fold increase in the Km value for IPP and a 1,000-2,000-fold lower kcat/Km value than those of the wild-type, although the Km values for FPP showed about no significant changes. In addition, the W224A mutant as to Region V showed 6-fold and 14-fold increased Km values for IPP and FPP, respectively, and 100-250-fold decreased kcat/Km values as compared to those of the wild-type. These results suggested that these conserved aromatic residues play important roles in the binding with both substrates, IPP and FPP, as well as the catalytic function of undecaprenyl diphosphate synthase.     

Interaction of carboxypeptidase A with carbamate and carbonate esters

The carbamate ester N-(phenoxycarbonyl)-L-phenylalanine binds well to carboxypeptidase A in the manner of peptide substrates. The ester exhibits linear competitive inhibition toward carboxypeptidase A catalyzed hydrolysis of the amide hippuryl-L-phenylalanine (Ki = 1.0 X 10(-3) M at pH 7.5) and linear noncompetitive inhibition toward hydrolysis of the specific ester substrate O-hippuryl-L-beta-phenyllactate (Ki = 1.4 X 10(-3) M at pH 7.5). Linear inhibition shows that only one molecule of inhibitor is bound per active site at pH 7.5. The hydrolysis of the carbamate ester is not affected by the presence of 10(-8)-10(-9) M enzyme (the concentrations employed in inhibition experiments), but at an enzyme concentration of 3 X 10(-6) M catalysis can be detected. The value of kcat at 30 degrees C, mu = 0.5 M, and pH 7.45 is 0.25 s-1, and Km is 1.5 X 10(-3) M. The near identity of Km and Ki shows that Km is a dissociation constant. Substrate inhibition can be detected at pH less than 7 but not at pH values above 7, which suggests that a conformational change is occurring near that pH. The analogous carbonate ester O-(phenoxycarbonyl)-L-beta-phenyllactic acid is also a substrate for the enzyme. The Km is pH independent from pH 6.5 to 9 and has the value of 7.6 X 10(-5) M in that pH region. The rate constant kcat is pH independent from pH 8 to 10 at 30 degrees C (mu = 0.5 M) with a limiting value of 1.60 s-1. Modification of the carboxyl group of glutamic acid-270 to the methoxyamide strongly inhibits the hydrolysis of O-(phenoxycarbonyl)-L-beta-phenyllactic acid. Binding of beta-phenyllactate esters and phenylalanine amides must occur in different subsites, but the ratios of kcat and kcat/Km for the structural change from hippuryl to phenoxy in each series are closely similar, which suggests that the rate-determining steps are mechanistically similar.     

Barnase has subsites that give rise to large rate enhancements.

Barnase is found to have a series of subsites for binding its substrates that confers large rate enhancements. Ribonucleotide substrates of the type Zp0Gp1Xp2Y have been synthesized, where p is phosphate, X, Y, and Z are nucleosides, and G is guanosine. G occupies the primary specificity site. The most important subsite is for p2, followed by that for Y. There appears to be no subsite for the Z or p0 positions. Occupation of the subsite for p2 gives rise to a 1000-fold increase in kcat/Km, composed of a 100-fold increase in kcat and a 10-fold decrease in Km. The Y subsite gives rise to further 20-fold increase in kcat/Km. Rates approaching diffusion control for kcat/Km are observed. kcat for the dinucleotide monophosphate GpU = 0.55 s-1, and Km = 240 microM; this compares with 53 s-1 and 20 microM for GpUp, and 3.3 x 10(3) s-1 and 17 microM for GpApA (the best substrate tested). Cleavage occurs at the 3'-phosphate of guanosine in all cases. There are differences in base specificity at the two subsites for X and Y downstream of the scissile bond. The binding energies of different substrates have been analyzed using thermodynamic cycles. These show that the contributions of the X and Y sites are nonadditive.     

Kinetic study of carboxypeptidase P-catalyzed reaction. Pressure and temperature dependence of kinetic parameters

Detailed kinetic analyses of carboxypeptidase P-catalyzed reactions were carried out spectrophotometrically using 3-(2-furyl)acryloyl-acylated peptide substrates. The maximum kcat/Km was observed at around pH 3.5 for the synthetic peptide substrates. The kcat/Km value decreased with increasing pH, with an apparent pKa value of 4.43. However, the maximum kcat was observed at neutral pH (pH congruent to 6) and the pKa was 4.49. These apparently different pH profiles for kcat/Km and kcat of this enzyme were due to the decreasing Km value in the acid pH region. The pressure and temperature dependences of these kinetic parameters were also measured. N-Benzoylglycyl-L-phenyllactate (Bz-Gly-OPhLac) gave dependences similar to those of the peptide substrate, suggesting that there is no distinct difference in the catalytic mechanism between the peptide and the ester hydrolyses.     

Transition state model and mechanism of nucleophilic aromatic substitution reactions catalyzed by human glutathione S-transferase M1a-1a

An active site His107 residue distinguishes human glutathione S-transferase hGSTM1-1 from other mammalian Mu-class GSTs. The crystal structure of hGSTM1a-1a with bound glutathione (GSH) was solved to 1.9 A resolution, and site-directed mutagenesis supports the conclusion that a proton transfer occurs in which bound water at the catalytic site acts as a primary proton acceptor from the GSH thiol group to transfer the proton to His107. The structure of the second substrate-binding site (H-site) was determined from hGSTM1a-1a complexed with 1-glutathionyl-2,4-dinitrobenzene (GS-DNB) formed by a reaction in the crystal between GSH and 1-chloro-2,4-dinitrobenzene (CDNB). In that structure, the GSH-binding site (G-site) is occupied by the GSH moiety of the product in the same configuration as that of the enzyme-GSH complex, and the dinitrobenzene ring is anchored between the side chains of Tyr6, Leu12, His107, Met108, and Tyr115. This orientation suggested a distinct transition state that was substantiated from the structure of hGSTM1a-1a complexed with transition state analogue 1-S-(glutathionyl)-2,4,6-trinitrocyclohexadienate (Meisenheimer complex). Kinetic data for GSTM1a-1a indicate that kcat(CDNB) for the reaction is more than 3 times greater than kcat(FDNB), even though the nonenzymatic second-order rate constant is more than 50-fold greater for 1-fluoro-2,4-dinitrobenzene (FDNB), and the product is the same for both substrates. In addition, Km(FDNB) is about 20 times less than Km(CDNB). The results are consistent with a mechanism in which the formation of the transition state is rate-limiting in the nucleophilic aromatic substitution reactions. Data obtained with active-site mutants support transition states in which Tyr115, Tyr6, and His107 side chains are involved in the stabilization of the Meisenheimer complex via interactions with the ortho nitro group of CDNB or FDNB and provide insight into the means by which GSTs adapt to accommodate different substrates.     

Human aldo-keto reductases: Function, gene regulation, and single nucleotide polymorphisms

Aldo-keto reductases (AKRs) are a superfamily of NAD(P)H linked oxidoreductases that are generally monomeric 34-37kDa proteins present in all phyla. The superfamily consists of 15 families, which contains 151 members (www.med.upenn.edu/akr). Thirteen human AKRs exist that use endogenous substrates (sugar and lipid aldehydes, prostaglandins, retinals and steroid hormones), and in many instances they regulate nuclear receptor signaling. Exogenous substrates include metabolites implicated in chemical carcinogenesis: NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), polycyclic aromatic hydrocarbon trans-dihydrodiols, and aflatoxin dialdehyde. Promoter analysis of the human genes identifies common elements involved in their regulation which include osmotic response elements, anti-oxidant response elements, xenobiotic response elements, AP-1 sites and steroid response elements. The human AKRs are highly polymorphic, and in some instances single nucleotide polymorphisms (SNPs) of high penetrance exist. This suggests that there will be inter-individual variation in endogenous and xenobiotic metabolism which in turn affect susceptibility to nuclear receptor signaling and chemical carcinogenesis.     

Kinetic independence of the subunits of cytosolic glutathione transferase from the rat.

The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.     

Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.     

Trypsin catalyzed hydrolysis of new chromogenic arginine substrates

Trypsin catalyzed hydrolysis of seven new chromogenic arginine substrates, N alpha-benzyloxycarbonyl-L-arginine-3-nitro-5X-anilide (X = H, CF3, SO2CH3, F, Cl, Br and I) were studied. These substrates are suitable for studying electronic effects on trypsin activity. The Km and kcat values for the hydrolysis of each substrate were determined and found to differ significantly for the various substrates. The Hammett plot of the catalytic rate constants gave a straight line with a negative rho value (-0.82) thus indicating that electron withdrawing substituents retard the trypsin catalyzed hydrolysis of the new anilide substrates.     

Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study

Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism: (formula; see text) Under pre-steady-state conditions, where [E0] much greater than [S0], the values of the dissociation constant of the E X S complex (Ks = k-1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] much greater than [E0], the values of kcat and Km have been obtained over the same pH range. The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 +/- 0.10. The values of Ks and Km are pH independent. The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.beta-trypsin and b.alpha-chymotrypsin. For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0. The different catalytic behaviours of p.p. elastase, b.beta-trypsin and b.alpha-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.     

Gene structure, purification and characterization of DNA polymerase beta from Xiphophorus maculatus

Cloning of the Xiphophorus maculatus Polbeta gene and overexpression of the recombinant Polbeta protein has been performed. The organization of the XiphPolbeta introns and exons, including intron-exon boundaries, have been assigned and were found to be similar to that for human Polbeta with identical exon sizes except for exon XII coding for an additional two amino acid residues in Xiphophorus. The cDNA sequence encoding the 337-amino acid X. maculatus DNA polymerase beta (Polbeta) protein was subcloned into the Escherichia coli expression plasmid pET. Induction of transformed E. coli cells resulted in the high-level expression of soluble recombinant Polbeta, which catalyzed DNA synthesis on template-primer substrates. The steady-state Michaelis constants (Km) and catalytic efficiencies (kcat/Km) of the recombinant XiphPolbeta for nucleotide insertion opposite single-nucleotide gap DNA substrates were measured and compared with previously published values for recombinant human Polbeta. Steady-state in vitro Km and kcat/Km values for correct nucleotide insertion by XiphPolbeta and human Polbeta were similar, although the recombinant Xiphophorus protein exhibited 2.5-7-fold higher catalytic efficiencies for dGTP and dCTP insertion versus human Polbeta. In contrast, the recombinant XiphPolbeta displayed significantly lower fidelities than human Polbeta for dNTP insertion opposite a single-nucleotide gap at 37 degrees C.     

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors.

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.