Conformation of the active site of thiolsubtilisin: reaction with specific chloromethyl ketones and arylacryloylimidazoles.

The conformation of the active site of thiolsubtilisin, prepared from subtilisin by transformation of the active site Ser to Cys, was compared with that of subtilisin by kinetic and spectroscopic methods. Carbobenzyloxy-L-alanylglycyl-L-phenylalanine chloromethyl ketone inhibited thiolsubtilisin approximately 10(2) times faster than subtilisin; alkylation occurred at the sulfhydryl rather than the imidazolyl group of the active site. pH dependence of the inhibition is different from that of the reaction between a simple thiol with haloacetamide. Furthermore, several native chromophoric arylacryloyl-thiolsubtilisins and arylacryloyl-subtilisins showed similar red shifts when compared with their denatured forms. The rate of deacylation of arylacryloyl-thiolsubtilisins was faster than (or of the same order of magnitude as) the deacylation rate of the analogous arylacryloyl-subtilisins in 30% dioxane (v/v), pH 5--10. The deacylation rate--pH profiles of these arylacryloyl-thiolsubtilisins in 30% dioxane all give pK values of 7.7 which is identical with the pK in the deacylation of acyl-subtilisins. These facts strongly suggest that the active-site conformation remains intact on conversion from subtilisin to thiolsubtilisin. The low esterase and peptidase activities of thiolsubtilisin are most likely due to the relatively low basicity of -SH (compared with -OH).     

Kinetics of subtilisin and thiolsubtilisin

Subtilisin is a bacterial serine protease with a broad specificity in the S1 subsite. It has been very extensively studied using a variety of kinetic and physical techniques. A chemical derivative, thiolsubtilisin, has been subjected to similar studies in order to analyze the effects of the OH to SH conversion on enzyme activity. The native structure of thiolsubtilisin is indicated by a variety of physical techniques. Oligopeptides bind nearly equally well to both enzymes, and a peptide chloromethylketone is much more reactive to thiolsubtilisin than to subtilisin. Both enzymes have a similar level of activity towards activated nonspecific amides and esters. However, thiolsubtilisin is inactive towards highly specific peptide amides and esters. Thiolsubtilisin also does not show good binding to boronic and arsonic acids. The observation that these transition state analog inhibitors bind poorly to thiolsubtilisin while other compounds bind nearly equally well to both enzymes suggests that thiolsubtilisin may not be able to stabilize the transition state during acylation by specific substrates.     

Interaction of thiolsubtilisin with Streptomyces subtilisin inhibitor, SSI.

Subtilisin BPN' was chemically converted to thiolsubtilisin and the interaction of this modified enzyme with Streptomyces subtilisin inhibitor (SSI) was examined. SSI competitively inhibited the esterolytic activity of thiolsubtilisin toward p-nitrophenyl acetate with a K1 value of 1.3 X 10(-5) M at pH 7.5 Spectrophotometric analysis of the interaction between SSI and the modified enzyme yielded a Kd value of 4 X 10(-5) M at pH 9.7. These values are about 10(5)-fold greater than the Kd value (less than 10(-9) M at pH 7.5) for the native enzyme. This indicates that the small change in the active site structure of subtilisin (Ser221 to Cys221) leads to a considerable decrease in the binding affinity (by about 6-7 kcal/mol) to SSI.     

A comparison of dogfish and bovine chymotrypsins

Bovine and dogfish chymotrypsins were compared to determine if chymotrypsin from a poikilothermic organism (spiny dogfish (Squalus acanthias] adapted to low temperatures possessed catalytic properties different from those of the same enzyme from a warm-blooded animal. An improved procedure was developed for isolating dogfish pancreatic chymotrypsin. The least hydrophobic and smallest substrate used, p-nitrophenyl acetate, had similar enthalpies of association (delta Ha) with both enzymes, whereas larger, more hydrophobic substrates had delta Ha values that were of opposite sign for the two enzymes. As the temperature increased, the association constants (1/Ks) for p-nitrophenyl valerate and p-nitrophenyltrimethyl acetate increased for dogfish chymotrypsin and decreased for bovine chymotrypsin, while the free energies of association (delta Ga) remained relatively constant. Acylation of chymotrypsin was 1.5-2.5 times slower in the dogfish enzyme than in the bovine enzyme except below 15 degrees C with p-nitrophenyltrimethyl acetate. delta H++ for acylation by p-nitrophenyltrimethyl acetate were 2.0 kcal/mol for the dogfish enzyme and 10.2 kcal/mol for the bovine, whereas delta H++ values were only slightly lower in the dogfish enzyme for the other two substrates. For all substrates, the deacylation rate constant (kcat) was greater with dogfish chymotrypsin than bovine. However, the free energies of activation (delta G++) for deacylation were nearly equal between the two enzymes for each of the substrates.     

Beta-lactamases as fully efficient enzymes. Determination of all the rate constants in the acyl-enzyme mechanism.

The rate constants for both acylation and deacylation of beta-lactamase PC1 from Staphylococcus aureus and the RTEM beta-lactamase from Escherichia coli were determined by the acid-quench method [Martin & Waley (1988) Biochem. J. 254, 923-925] with several good substrates, and, for a wider range of substrates, of beta-lactamase I from Bacillus cereus. The values of the acylation and deacylation rate constants for benzylpenicillin were approximately the same (i.e. differing by no more than 2-fold) for each enzyme. The variation of kcat./Km for benzylpenicillin with the viscosity of the medium was used to obtain values for all four rate constants in the acyl-enzyme mechanism for all three enzymes. The reaction is partly diffusion-controlled, and the rate constant for the dissociation of the enzyme-substrate complex has approximately the same value as the rate constants for acylation and deacylation. Thus all three first-order rate constants have comparable values. Here there is no single rate-determining step for beta-lactamase action. This is taken to be a sign of a fully efficient enzyme.     

Kinetic behaviour of alpha-chymotrypsin in reverse micelles. A stopped-flow study.

At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of alpha-chymotrypsin have been studied in reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. Pre-steady state and steady-state kinetic constants, in water and in reverse micelles, have been determined by stopped-flow spectrophotometry for the hydrolysis of two substrates, namely acetyl-L-tryptophan-p-nitrophenyl ester and p-nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k2) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kcat) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.     

Specificity of thrombin: evidence for selectivity in acylation rather than binding for p-nitrophenyl alpha-amino-p-toluate.

The lysyl ester analogue p-nitrophenyl alpha-amino-p-toluate hydrobromide was synthesized, and its reactions with thrombin, trypsin, and plasmin were studied by stopped-flow and conventional methods. Kinetic parameters were compared with those determined for the arginyl ester analogue, p-nitrophenyl p-guanidinobenzoate hydrochloride, with these enzymes. By following nitrophenol release or proflavin absorption changes in the stopped-flow spectrophotometer, the constants Ks (enzyme-substrate binding), k2 (acylation), and k3 (deacylation) were determined. The major findings were: (1) Ks values were similar regardless of the substrate or the enzyme; (2) k3 was approximately the same for the reaction of the lysyl ester analogue with any enzyme; (3) k2 for the lysyl ester analogue was 1100 times greater with trypsin than with thrombin; and (4) k2 with thrombin was 60 times greater for the arginyl than for the lysyl ester analogue. The results suggest that the limited cleavage of lysyl bonds by thrombin is due in part to restricted acylation rather than substrate binding. The active site of thrombin, compared with that of trypsin, appears to have a more stringent requirement for the spatial relationship between the cationic group and the bond cleaved in substrates.     

Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEM(pUC19) beta-lactamase from Escherichia coli.

We investigated the effects of mutations at positions 164 and 179 of the TEM(pUC19) beta-lactamase on turnover of substrates. The direct consequence of some mutations at these sites is that clinically important expanded-spectrum beta-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A beta-lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum beta-lactams by their enhanced ability to turn over these antibiotic substrates. Four of these mutant variants, Arg(164) --> Asn, Arg(164) --> Ser, Asp(179) --> Asn, and Asp(179) --> Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded-spectrum beta-lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K(s)) invariably increased. Computation of the microscopic rate constants (k(2) and k(3)) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k(3)) or that neither the acylation nor deacylation became the sole rate-limiting step. In a few instances, the rate constants for both the acylation (k(2)) and deacylation (k(3)) of the extended-spectrum beta-lactamase were enhanced. These results were investigated further by molecular modeling experiments, using the crystal structure of the TEM(pUC19) beta-lactamase. Our results indicated that severe steric interactions between the large 7beta functionalities of the expanded-spectrum beta-lactams and the Omega-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity.     

Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)- [NAD(P)-] dependent aldehyde dehydrogenases share a number of conserved amino acid residues, several of which are directly implicated in catalysis. In the present study, the role of Glu-268 from nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans was investigated. Its substitution by Ala resulted in a k(cat) decrease by 3 orders of magnitude. Pre-steady-state analysis showed that, for both the wild-type and E268A GAPNs, the rate-limiting step of the reaction is associated with deacylation. The pH dependence of the rate of acylation of wild-type GAPN is characterized by the contributions of distinct enzyme protonic species with two pK(a)s of 6.2 and 7.5. Substitution of Glu-268 by Ala resulted in a monosigmoidal pH dependence of the rate constant of acylation with a pK(a) of 6.2, which suggested the assignment of pK(a) 7.5 to Glu-268. Moreover, the E268A substitution did not significantly affect the efficiency of acylation of GAPN, showing that Glu-268 is not critically involved in the acylation, which includes Cys-302 nucleophilic activation and hydride transfer. On the contrary, the drastic decrease of the steady-state rate constant for the E268A GAPN demonstrated the essential role of Glu-268 in the deacylation. At basic pH, the solvent isotope effect of 2.3, characterized by a unique pK(a) of 7.7, and the linearity of the proton inventory showed that the rate-limiting process for deacylation is associated with the hydrolysis step and suggested that the glutamate form of Glu-268 acts as a base catalyst in this process. Surprisingly, the double-sigmoidal form of the pH-steady-state rate constant profile, characterized by pK(a) values of 6.1 and 7.4, revealed the high efficiency of the deacylation even at pH lower than 7.4. Therefore, we propose that the major role of Glu-268 is to promote deacylation through activation and orientation of the attacking water molecule, and in addition to act as a base catalyst at basic pH. From these results in relation to those recently described [Marchal, S., and Branlant, G. (1999) Biochemistry 38, 12950-12958], a scenario for the chemical catalysis of GAPN is proposed.     

pH effects in plasmin-catalysed hydrolysis of alpha-N-benzoyl-L-arginine compounds.

The steady-state kinetics of plasmin (EC 3.4.21.7) catalysed reactions with some alpha-N-benzoyl-L-arginine compounds is investigated in the pH range 5.8--9.0. The results are interpreted in terms of a three-step mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. Alpha-N-Benzoyl-L-arginine methyl ester and ethyl ester show the same pH behaviour. The kinetic parameter kc/Km is influenced by two groups with pK values of 6.5 and 8.4, respectively. kc is affected only by the group with pK equal to 6.5 and Km only by the group with pK equal to 8.4. It is suggested that the group with pK equal to 6.5 is the 1-chloro-3-tosyl-amido-7-amino-2-heptanone-sensitive histidine residue in the active site and that the group with pK equal to 8.4 is perhaps the alpha-amino group of the N-terminus in analogy to trypsin and chymotrypsin. alpha-N-Benzoyl-L-arginine amide is not hydrolysed by plasmin, but proves to be a competitive inhibitor, Ki = 12.8 +/- 1.8 mM, pH = 7.8. Also the product alpha-N-benzoyl-L-arginine is a competitive inhibitor, Ki = 26 +/- 3.1 mM, pH = 7.8. Estimates of individual rate constants are compared with similar trypsin data.     

Lysine-73 is involved in the acylation and deacylation of beta-lactamase

Lysine 73 is a conserved active-site residue in the class A beta-lactamases, as well as other members of the serine penicillin-sensitive enzyme family; its role in catalysis remains controversial and uncertain. Mutation of Lys73 to alanine in the beta-lactamase from Bacillus licheniformis resulted in a substantial reduction in both turnover rate (k(cat)) and catalytic efficiency (k(cat)/K(m)), and a very significant shift in pK(1) to higher pH in the bell-shaped pH-rate profiles (k(cat)/K(m)) for several penicillin and cephalosporin substrates. The increase in pK(1) is consistent with the removal of the positive ammonium group of the lysine from the proximity of Glu166, to which the acid limb has been ascribed. The alkaline limb of the k(cat)/K(m) vs profiles is not shifted appreciably, as might have been expected if this limb reflected the ionization of Lys73 in the wild-type enzyme. The k(cat)/K(m) at the pH optimum for the mutant was down about 200-fold for penicillins and around 10(4) for cephalosporins, compared to the wild-type, suggesting significant differences in the mechanisms for catalysis of penicillins compared to cephalosporins. Burst kinetics were observed with several substrates assayed with K73A beta-lactamase, indicating an underlying branched-pathway kinetic scheme, and rate-limiting deacylation. FTIR analysis was used to determine whether acylation or deacylation was rate-limiting. In general, acylation was the rate-limiting step for cephalosporin substrates, whereas deacylation was rate-limiting for penicillin substrates. The results indicate that Lys73 plays an important role in both the acylation and deacylation steps of the catalytic mechanism. The effects of this mutation (K73A) indicate that Lys73 does not function as a general base in the catalytic mechanism of beta-lactamase. The existence of bell-shaped pH-rate profiles for the K73A variant suggests that Lys73 is not directly responsible for either limb in such plots. It is likely that both Glu166 and Lys73 are important to each other in terms of maintaining the optimum electrostatic environment for fully efficient catalytic activity to occur.     

The esterase activity of bovine carbonic anhydrase B above pH 9. Reversible and cooalent inhibition by acetozolamide.

The reversible complex between the metalloenzyme bovine carbonic anhydrase B and the sulfonamide inhibitor acetazolamide can be "frozen" irreversibly by the addition of a covalent bond between the methyl group of the inhibitor and the tau-nitrogen of histidine-64. In both cases the inhibited enzyme is inactive as an esterase toward p-nitrophenyl propionate at physiological pH but retains activity controlled by an ionization in the protein exhibiting a pK-a greater than 10. Similarly, both the covalently and reversibly inhibited enzymes in which the catalytically essential Zn(II) ion has been replaced with Co(II) display the same visible absorption spectrum which is invariant over the pH range from 5 to 12. The evidence therefore indicates that the position of the acetazolamide moiety in the active site is independent of both pH and the presence of the covalent bond to histidine-64. Moreover, when reversibly bound, this inhibitor has been shown to replace the water molecule (or hydroxide ion) known to occupy the fourth coordination position of the metal ion and frequently implicated in the catalytic mechanism of carbonic anhydrases. Thus, the activity exhibited by the inhibited enzymes and consequently the second rise observed in the pH rate profile of the native enzyme above pH 0 cannot reflect the ionization of such a water molecule in contrast to what has been postulated previously (Pocker, Y., and Storm, D. R. (1968) Biochemistry 7, 1202-1214). Displacement of the zinc-bound solvent molecule rather than the alkylation of histidine-64 is suggested, however, as the cause of the inactivation of the alkylated enzyme round neutrality. Taken together, the biphasic pH rate profile of native bovine carbonic anhydrase B as well as the activity retained by the alkylated enzyme above pH 9 are best described by a model in which two groups in the enzyme ionize independently, thereby raising the possibility that the high pH activity is controlled by an ionization outside the active site region of the enzyme. Above pH 9.5 the pK; for the reversible interaction between native carbonic anhydrase and acetazolamide falls off linearly with increasing pH. The slope of --1.56 suggests that, among other factors, more than one ionization is responsible for the descending limb of the pH-i-pH profile.     

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system: effects of water on kinetic parameters

The influence of water on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates to the enzyme while removing reaction products. In this system, interactions between the enzyme and nonreacting molecules are avoided, since no solvent is present, and it is thus more easy to assess the role of water. To this end, alcohol inhibition constant, substrates dissociation constants as well as acylation rate constant and ratio of acylation to deacylation rate constants have been determined as a function of water activity (a(w)). Data obtained highlight that n-propanol inhibition constant and dissociation constant of methyl propionate are a lot affected by a(w) variations whereas water has no significant effect on the catalytic acylation step nor on the ratio of acylation to deacylation rate constants. These results suggest the water-independent character of the transition step.     

Influence of hydrophobic and steric effects in the acyl group on acylation of alpha-chymotrypsin by N-acylimidazoles

The second-order rate constants k2/Km for acylation of alpha-chymotrypsin by a series of N-acylimidazole derivatives of aliphatic carboxylic acids have been determined at 30 degrees C by proflavin displacement from the active site. With cyclohexyl-substituted N-acylimidazoles, the rate constants increase with increasing chain length of the acyl group; i.e., k2/Km is in the order cyclohexylcarbonyl less than cyclohexylacetyl less than beta-cyclohexylpropionyl. The latter substrate has k2/Km = 1.2 X 10(6) M-1 s-1 at pH 8.0, which appears to be a maximum value for N-acylimidazole substrates. A further increase in the chain length of the acyl group with (gamma-cyclohexylbutyryl)imidazole results in a decrease in k2/Km. Hydrophobic effects of the hydrocarbon acyl groups are of predominant importance with regard to the relative values of k2/Km for aliphatic N-acylimidazole substrates. There is a linear correlation of the logarithms of the rate constants at pH 8.0 with the hydrophobic substituent constants, pi, having a slope of 1.71 (r = 0.90). On the other hand, there is little apparent correlation with the Taft steric effect constants, Es. A four-parameter equation including both pi and Es improved the correlation only slightly [log (k2/Km) = 1.88 pi + 1.01 Es + C]. In contrast, steric effects as reflected in the Es constants are the major influence in acylation of the enzyme by corresponding p-nitrophenyl esters. There are very likely significant differences in transition-state structure with the two types of substrates.     

The pH dependency of bovine spleen cathepsin B-catalyzed transfer of N alpha-benzyloxycarbonyl-L-lysine from p-nitrophenol to water and dipeptide nucleophiles. Comparisons with papain

Cathepsin B has been shown to catalyze the transfer of the N alpha-benzyloxycarbonyl-L-lysyl residue from the corresponding p-nitrophenyl ester substrate to water and dipeptide nucleophiles. These reactions occurred through the formation of an acyl-enzyme intermediate. The pH dependency of the acylation and deacylation steps were determined from the increases in the maximum rate of appearance of p-nitrophenol on addition of glycylglycine or L-leucylglycine to the reaction. The second order acylation rate constant, kcat/Km was found to depend on the state of ionization of three groups in the enzyme having pKa values of 4.2, 5.5, and 8.6. Protonation of the group with pKa = 5.5 decreased but did not abolish enzymatic activity, resulting in the appearance of a second, active protonic form of the enzyme between pH 4.2 and pH 5.5. The first order rate constant for the hydrolysis of the acyl-enzyme intermediate was independent of pH between 4.0 and 7.5. In contrast, acyl group transfer from cathepsin B to glycylglycine and L-leucylglycine depended on a group with a pKa of about 4.5. These results are discussed in terms of possible structural and functional homologies between the active sites of cathepsin B and papain.     

Binding rates, O--S substitution effects, and the pH dependence of chymotrypsin reactions.

The pH dependence for acylation of alpha-chymotrypsin by N-acetyltryptophan p-nitrophenyl-, p-nitrothiophenyl-, ethyl-, and thiolethyl esters has been studied by the stopped-flow technique. Values for the acylation rate constant, k2, and the binding constant, KS, were obtained by using measurements of phenolate release, for the p-nitrophenyl esters, and proflavin displacement, for the ethyl esters. The oxygen esters tested have slightly higher k2 values, and substantially higher KS values relative to the analogous thiol esters. Whereas k2/KS for the thiolethyl ester is higher than that for the analogous oxygen ester, the k2/KS values for oxy- and thio-p-nitrophenyl esters are nearly identical. These data are interpreted to indicate rate-determining formation of a tetrahedral intermediate in acylation of alpha-chymotrypsin by p-nitrophenyl esters, and rate-determining breakdown of such an intermediate in the case of the ethyl esters. It is also concluded that the oxygen to sulfur substitution causes a substantial increase in the proportion of nonproductive binding in these substrates. pH dependent k2 and KS values were used to calculate values for k1 and k-1, the binding and debinding rate constants for the two p-nitrophenyl compounds. This is the first such calculation based on experimentally determined acylation rate constants.     

Investigation of the active center of rat pancreatic elastase

We have isolated rat pancreatic elastase I (EC 3.4.21.36) using a fast two-step procedure and we have investigated its active center with p-nitroanilide substrates and trifluoroacetylated inhibitors. These ligands were also used to probe porcine pancreatic elastase I whose amino acid sequence is 84% homologous to rat pancreatic elastase I as reported by MacDonald, et al. (Biochemistry 21, (1982) 1453-1463). Both proteinases exhibited non-Michaelian kinetics for substrates composed of three or four residues: substrate inhibition was observed for most enzyme substrate pairs, but with Ala3-p-nitroanilide, rat elastase showed substrate inhibition, whereas porcine elastase exhibited substrate activation. With most of the longer substrates, Michaelian kinetics were observed. The kcat/Km ratio was used to compare the catalytic efficiency of the two elastases on the different substrates. For both elastases, occupancy of subsite S4 was a prerequisite for efficient catalysis, occupancy of subsite S5 further increased the catalytic efficiency, P2 proline favored catalysis and P1 valine had an unfavorable effect. Rat elastase has probably one more subsite (S6) than its porcine counterpart. The rate-limiting step for the hydrolysis of N-succinyl-Ala3-p-nitroanilide by rat elastase was essentially acylation, whereas both acylation and deacylation rate constants participated in the turnover of this substrate by porcine elastase. For both enzymes, trifluoroacetylated peptides were much better inhibitors than acetylated peptides and trifluoroacetyldipeptide anilides were more potent than trifluoroacetyltripeptide anilides. A number of quantitative differences were found, however, and with one exception, trifluoroacetylated inhibitors were less efficient with rat elastase than with the porcine enzyme.     

A catalytic triad is responsible for acid-base chemistry in the Ascaris suum NAD-malic enzyme

The pH dependence of kinetic parameters of several active site mutants of the Ascaris suum NAD-malic enzyme was investigated to determine the role of amino acid residues likely involved in catalysis on the basis of three-dimensional structures of malic enzyme. Lysine 199 is positioned to act as the general base that accepts a proton from the 2-hydroxyl of malate during the hydride transfer step. The pH dependence of V/K(malate) for the K199R mutant enzyme reveals a pK of 5.3 for an enzymatic group required to be unprotonated for activity and a second pK of 6.3 that leads to a 10-fold loss in activity above the pK of 6.3 to a new constant value up to pH 10. The V profile for K199R is pH independent from pH 5.5 to pH 10 and decreases below a pK of 4.9. Tyrosine 126 is positioned to act as the general acid that donates a proton to the enolpyruvate intermediate to form pyruvate. The pH dependence of V/K(malate) for the Y126F mutant is qualitatively similar to K199R, with a requirement for a group to be unprotonated for activity with a pK of 5.6 and a partial activity loss of about 3-fold above a pK of 6.7 to a new constant value. The Y126F mutant enzyme is about 60000-fold less active than the wild-type enzyme. In contrast to K199R, the V rate profile for Y126F also shows a partial activity loss above pH 6.6. The wild-type pH profiles were reinvestigated in light of the discovery of the partial activity change for the mutant enzymes. The wild-type V/K(malate) pH-rate profile exhibits the requirement for a group to be unprotonated for catalysis with a pK of 5.6 and also shows the partial activity loss above a pK of 6.4. The wild-type V pH-rate profile decreases below a pK of 5.2 and is pH independent from pH 5.5 to pH 10. Aspartate 294 is within hydrogen-bonding distance to K199 in the open and closed forms of malic enzyme. D294A is about 13000-fold less active than the wild-type enzyme, and the pH-rate profile for V/K(malate) indicates the mutant is only active above pH 9. The data suggest that the pK present at about pH 5.6 in all of the pH profiles represents D294, and during catalysis D294 accepts a proton from K199 to allow K199 to act as a general base in the reaction. The pK for the general acid in the reaction is not observed, consistent with rapid tautomerization of enolpyruvate. No other ionizable group in the active site is likely responsible for the partial activity change observed in the pH profiles, and thus the group responsible is probably remote from the active site and the effect on activity is transmitted through the protein by a conformational change.     

Modification of isoleucine-16 acetylated delta-chymotrypsin.

Activation of acetylated chymotrypsinogen with trypsin leads to catalytically active acetylated delta-chymotrypsin containing NH2-terminal isoleucine. The importance of the cationic terminus to the control of the active conformation of acetylated delta-chymotrypsin has been demonstrated (Oppenheimer, H. L., Labouesse, B., and Hess, G. P. (1966) J. Biol. Chem. 241, 2720). Later studies appeared to suggest that the modification of isoleucine-16 of delta-chymotrypsin is not accompanied by the loss of catalytic activity as measured by the hydrolysis of N-acetyl-L-tyrosine ethyl ester (Agarwal, S. P., Martin, C. J., Blair, T. T., and Marini, M.A. (1971)Biochem. Biophys. Res. Commun. 43, 510; Blair, T. T., Marini, M. A., Agarwal, S. P., and Martin, C. J. (1971) FEBS Lett. 1486) or by the loss of active site content (Ghelis, C., Garel, J. R., and Labouesse, J. (1970) Biochemistry 9, 3902). In the present studies, controlled acetylation of the terminal alpha-aminogroup of acetylated delta-chymotrypsin with acetic anhydride led to a progressive loss of active sites of the enzyme. Determination of the catalytic and kinetic properties of the modified enzyme with the specific ester substrate N-acetyl-L-tyrosine ethyl ester or the nonspecific substrates p-nitrophenyl acetate and cinnamyol imidazole gave nearly identical results. With N-acetyl-L-tyrosine ethyl ester as substrate, the Km (app) values for acetylated delta-chymotrypsin (1.0 plus or minus 0.1 mM) and the modified enzyme (0.67 plus or minus 0.05 mM) are nearly identical and the kcat value is reduced to about 25% in the latter enzyme species. This value correlates well with about 20% of the active sites in this enzyme as measured by the rapid initial liberation of p-nitrophenol. With p-nitrophenyl acetate as substrate, the acylation rate constants (0.13 plus or minus 0.04 s(-1) at pH 6.0, 25 degrees, in 3.3% acetonitrile) and the deacylation rate constants (0.01 s(-1) at pH 8.5, 25 degrees, in 3.3% acetonitrile) are identical for the acetyl isoleucine-16 and the isoleucine-16 enzymes. Furthermore, the residual enzyme activity could be correlated well with the residual NH2-terminal isoleucine content and with the moles of [1--14C]acetyl groups incorporated per mol of the enzyme. The activity associated with the modified enzyme can be attributed to the enzyme species in which isoleucine-16 of acetylated delta-chymotrypsin is not acetylated. These data are in general agreement with the studies of Ghelis et al. (1970) but are in disagreement with the results of Blair et al. (1971) and of Agarwal et al. (1971) and confirm the hypothesis that the final conformation of acetylated delta-chymotrypsin containing an acetylated NH2 terminus is catalytically inactive and resembles acetylated zymogen in many of its physical properties.     

Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli

Penicillin-binding proteins (PBPs) and beta-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a dd-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced gamma-thialysine at each of these positions. The pH dependence of kcat/Km and of kcat for the wild-type PBP 5 and for the two gamma-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.