Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis.

The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.     

Enzyme-induced strain/distortion in the ground-state ES complex in beta-lactamase catalysis revealed by FTIR

Class A beta-lactamases hydrolyze penicillins and other beta-lactams via an acyl-enzyme catalytic mechanism. Ser70 is the active site nucleophile. By constructing the S70A mutant, which is unable to form the acyl-enzyme intermediate, it was possible to make stable ES complexes with various substrates. The stability of such Michaelis complexes permitted acquisition of their infrared spectra. Comparison of the beta-lactam carbonyl stretch frequency (nu(CO)) in the free and enzyme-bound substrate revealed an average decrease of 13 cm(-)(1), indicating substantial strain/distortion of the lactam carbonyl when bound in the ES complex. Interestingly, regardless of the frequency of the C=O stretch in the free substrate, when complexed to Bacillus licheniformis beta-lactamase, the frequency was always 1755 +/- 2 cm(-)(1). This suggests the active site environment induces a similar conformation of the beta-lactam in all substrates when bound to the enzyme. Using deuterium substitution, it was shown that the "oxyanion hole", which involves hydrogen bonding to two backbone amides, is the major source of the enzyme-induced strain/distortion. The very weak catalytic activity of the S70A beta-lactamase suggests enzyme-facilitated hydrolysis due to substrate distortion on binding to the enzyme. Thus the binding of the substrate in the active site induces substantial strain and distortion that contribute significantly to the overall rate enhancement in beta-lactamase catalysis.     

Spectral observation of an acyl-enzyme intermediate of lipoprotein lipase

There have been several studies indicating that hydrolysis reactions of fatty acid esters catalyzed by lipases proceed through an acyl-enzyme intermediate typical of serine proteases. In particular, one careful kinetic study with the physiologically important enzyme lipoprotein lipase (LPL) is consistent with rate-limiting deacylation of such an intermediate. To observe the spectrum of acyl-enzyme and study the mechanism of LPL-catalyzed hydrolysis of substrate, we have used a variety of furylacryloyl substrates including 1,2-dipalmitoyl-3-[(beta-2-furylacryloyl)triacyl]glyceride (DPFATG) to study the intermediates formed during the hydrolysis reaction catalyzed by the enzyme. After isolation and characterization of the molecular weight of adipose LPL, we determined its extinction coefficient at 280 nm to quantitate the formation of any acyl-enzyme intermediate formed during substrate hydrolysis. We observed an intermediate at low pH during the enzyme-catalyzed hydrolysis of (furylacryloyl)imidazole. This intermediate builds early in the reaction when a substantial amount of substrate has hydrolyzed but no product, furylacrylate, has been formed. The acyl-enzyme has a lambda max = 305 nm and a molar extinction coefficient of 22,600 M-1 cm-1; these parameters are similar to those for furylacryloyl esters including the serine ester. These data provide the first spectral evidence for a serine acyl-enzyme in lipase-catalyzed reactions. The LPL hydrolysis reaction is base catalyzed, exhibiting two pKa values; the more acidic of these is 6.5, consistent with base catalysis by histidine. The biphasic rates for substrate disappearance or product appearance and the absence of leaving group effect indicate that deacylation of intermediate is rate limiting.     

Crystal structure of Escherichia coli penicillin-binding protein 5 bound to a tripeptide boronic acid inhibitor: a role for Ser-110 in deacylation

Penicillin-binding protein 5 (PBP 5) from Escherichia coli is a well-characterized d-alanine carboxypeptidase that serves as a prototypical enzyme to elucidate the structure, function, and catalytic mechanism of PBPs. A comprehensive understanding of the catalytic mechanism underlying d-alanine carboxypeptidation and antibiotic binding has proven elusive. In this study, we report the crystal structure at 1.6 A resolution of PBP 5 in complex with a substrate-like peptide boronic acid, which was designed to resemble the transition-state intermediate during the deacylation step of the enzyme-catalyzed reaction with peptide substrates. In the structure of the complex, the boron atom is covalently attached to Ser-44, which in turn is within hydrogen-bonding distance to Lys-47. This arrangement further supports the assignment of Lys-47 as the general base that activates Ser-44 during acylation. One of the two hydroxyls in the boronyl center (O2) is held by the oxyanion hole comprising the amides of Ser-44 and His-216, while the other hydroxyl (O3), which is analogous to the nucleophilic water for hydrolysis of the acyl-enzyme intermediate, is solvated by a water molecule that bridges to Ser-110. Lys-47 is not well-positioned to act as the catalytic base in the deacylation reaction. Instead, these data suggest a mechanism of catalysis for deacylation that uses a hydrogen-bonding network, involving Lys-213, Ser-110, and a bridging water molecule, to polarize the hydrolytic water molecule.     

Mechanism of inhibition of RTEM-2 beta-lactamase by cephamycins: relative importance of the 7 alpha-methoxy group and the 3' leaving group

Cefoxitin is a poor substrate of many beta-lactamases, including the RTEM-2 enzyme. Fisher and co-workers [Fisher, J., Belasco, J. G., Khosla, S., & Knowles, J. R. (1980) Biochemistry 19, 2895-2901] showed that the reaction between cefoxitin and RTEM-2 beta-lactamase yielded a moderately stable acyl-enzyme whose hydrolysis was rate-determining to turnover at saturation. The present work shows first that the covalently bound substrate in this acyl-enzyme has a 5-exo-methylene-1,3-thiazine structure, i.e., that the good (carbamoyloxy) 3' leaving group of cefoxitin has been eliminated in formation of the acyl-enzyme. Such an elimination has recently been shown in another case to yield an acyl-beta-lactamase inert to hydrolysis [Faraci, W. S., & Pratt, R. F. (1985) Biochemistry 24, 903-910]. Thus the cefoxitin molecule has two potential sources of beta-lactamase resistance, the 7 alpha-methoxy group and the good 3' leaving group. That the latter is important in the present example is shown by the fact that with analogous substrates where no elimination occurs at the enzyme active site, such as 3'-de(carbamoyloxy)cefoxitin and 3'-decarbamoylcefoxitin, no inert acyl-enzyme accumulates. An analysis of the relevant rate constants shows that the 7 alpha-methoxy group weakens noncovalent binding and slows down both acylation and deacylation rates, but with major effect in the acylation rate, while elimination of the 3' leaving group affects deacylation only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman evidence for substrate reorginization in the active site of papain.

Resonance Raman spectra were obtained for the acylenzyme 4-dimethylamino-3-nitro(alpha-benzamido)cinnamoyl-papain prepared using the chromophoric substrate methyl 4-dimethylamino-3-nitro(alpha-benzamido)cinnamate. These spectra contained vibrational spectral data of the acyl residue while covalently attached to the active site and could be used to follow directly acylation and deacylation kinetics. Spectra were obtained at pH values ranging from those where the acyl-enzyme is relatively stable (pH 3.0, tau 1/2 congruent to 800 s) to those where it is relatively unstable (pH 9.2, tau 1/2 congruent to 223 s). Throughout this range acyl-enzyme spectra differed completely from that of the free substrate or the product (4-dimethylamino-3-nitro(alpha-benzamido)cinnamic acid) indicating that a structural change occurred on combination with the active site. The spectra are consistent with rearrangement of the alpha-benzamido group in the bound substrate, -NH--C(==O)Ph becoming --N==C(--OX)Ph, where the bonding to oxygen is unknown. Superimposed on these large differences, small changes in acyl-enzyme spectra also occurred as pH was raised to decrease the half-life. All of the above spectral perturbations are consistent with a structural change in the acyl-enzyme which precedes the rate-determining step in deacylation. Thus, deacylation proceeds from an acyl residue structure differing from that of the substrate in solution. Upon acid denaturation the spectrum characteristic of the intermediate reverts to one closely resembling the substrate, demonstrating that a functioning active site is necessary to produce the observed differences. Spectra in D2O of native acyl-enzyme were identical with those in H2O, indicating that the observed differences in rate constant were not due to solvent-induced structural changes. Activated papain purified by crystallization or by affinity chromatography formed the acyl-enzyme. However, the kinetics of formation and deacylation differed between these materials, as did the spectral properties. Small differences in active-site structure are considered to be responsible for this effect, and it is suggested that such spectral perturbations may be useful in directly relating small differences in structure of the substrate in the active site with corresponding differences in kinetics.     

Relocation of the catalytic carboxylate group in class A beta-lactamase: the structure and function of the mutant enzyme Glu166-->Gln:Asn170-->Asp.

The hydrolysis of beta-lactam antibiotics by the serine-beta-lactamases proceeds via an acyl-enzyme intermediate. In the class A enzymes, a key catalytic residue, Glu166, activates a water molecule for nucleophilic attack on the acyl-enzyme intermediate. The active site architecture raises the possibility that the location of the catalytic carboxylate group may be shifted while still maintaining close proximity to the hydrolytic water molecule. A double mutant of the Staphylococcus aureus PC1 beta-lactamase, E166Q:N170D, was produced, with the carboxylate group shifted to position 170 of the polypeptide chain. A mutant protein, E166Q, without a carboxylate group and with abolished deacylation, was produced as a control. The kinetics of the two mutant proteins have been analyzed and the crystal structure of the double mutant protein has been determined. The kinetic data confirmed that deacylation was restored in E166Q:N170D beta-lactamase, albeit not to the level of the wild-type enzyme. In addition, the kinetics of the double mutant enzyme follows progressive inactivation, characterized by initial fast rates and final slower rates. The addition of ammonium sulfate increases the size of the initial burst, consistent with stabilization of the active form of the enzyme by salt. The crystal structure reveals that the overall fold of the E166Q:N170D enzyme is similar to that of native beta-lactamase. However, high crystallographic temperature factors are associated with the ohm-loop region and some of the side chains, including Asp170, are partially or completely disordered. The structure provides a rationale for the progressive inactivation of the Asp170-containing mutant, suggesting that the flexible ohm-loop may be readily perturbed by the substrate such that Asp170's carboxylate group is not always poised to facilitate hydrolysis.     

Kinetic studies on the mechanism of the penicillin amidase-catalysed synthesis of ampicillin and benzylpenicillin

Hydrophobic protein chromatography was used to prepare homogeneous fractions of penicillin amidase (EC 3.5.1.11) from E. coli. The apparent ratios of the rate constants for the deacylation of the acyl-penicillin amidase formed in the hydrolysis of phenylacetylglycine or D-phenylglycine methyl ester, by H2O and 6-aminopenicillanic acid (6-APA), were determined at different concentrations of the latter compound. The ratios were obtained from direct measurements of the initial rates of formation of phenylacetic acid and benzylpenicillin or D-phenylglycine and ampicillin. For the semisynthesis of ampicillin as well as of benzylpenicillin the ratio was found to depend on the concentration of 6-APA. This was observed for heterogeneous and homogeneous enzyme preparations. These results show that 6-APA must be bound to the acyl-enzyme before the deacylation, yielding ampicillin and benzylpenicillin, occurs. The dissociation constant KN for the formation of the complex was estimated to be approximately 10mM. This mechanism in which acyl-enzyme with and without bound nucleophile is involved, is in agreement with the principle of microscopic reversibility. Both acyl-enzymes can be deacylated by H2O. The finding that there is a specific binding site for 6-APA adjacent to the binding site for the phenylacetyl-(D-phenylglycyl-) group in the active site of the enzyme is supported by the observation that 6-APA acts as a mixed inhibitor in the hydrolysis of D-phenylglycine methyl ester. The ionic strength dependence indicates that the binding site for 6-APA of the acyl-enzyme is positively charged.     

Effects of sulfhydryl reagents on the binding and release of penicillin G by D-alanine carboxypeptidase IA of Escherichia coli.

Purified D-alanine carboxypeptidase IA of Escherichia coli is inhibited by penicillin G and binds penicillin G reversibly. The binding of penicillin to the enzyme is relatively insensitive to sulfhydryl reagents, while release of penicillin from the enzyme is severely inhibited by these reagents. The inhibition of release parallels the inhibition of carboxypeptidase activity by the sulfhydryl reagents. In the presence of the sulfhydryl reagent p-chloromercuribenzoate, an acyl-enzyme intermediate, produced by the reaction of carboxypeptidase IA with diacetyl-L-lysyl-D-alanyl-D-alanine, accumulates and can be isolated. These results indicate that binding of penicillin to carboxypeptidase IA occurs by an acylation step of the carboxypeptidase reaction, while penicillin release occurs by a deacylation step of the reaction. Only the latter is inhibited by sulfhydryl reagents.     

Kinetics and mechanism of the serine beta-lactamase catalyzed hydrolysis of depsipeptides

Steady-state kinetic parameters have been determined for the hydrolysis of a series of acyclic depsipeptides (ester analogues of acyl-D-alanyl-D-alanine peptides) catalyzed by representative class C (Enterobacter cloacae P99) and class A (Bacillus cereus I, TEM-2, and Staphylococcus aureus PC1) beta-lactamases. The best of these substrates, and the one most used in this work, was m-[[(phenylacetyl)-glycyl]oxy]benzoic acid, whose rates of cleavage could be followed spectrophotometrically. The P99 enzyme also catalyzed the methanolysis of these substrates in aqueous methanol solutions. Quantitative evaluation of the effects of methanol on the kinetics of the competing hydrolysis and methanolysis reactions, and on the product distribution, supports a reaction mechanism involving an acyl-enzyme intermediate whose formation is rate-determining under conditions of substrate saturation. Consideration of the variation of these kinetic parameters with the structure of the depsipeptides and comparison with the analogous parameters for bicyclic beta-lactam substrates suggest that a variety of substrate binding modes exist on this enzyme. The class A enzymes, B. cereus beta-lactamase I and the TEM-2 beta-lactamase, catalyze depsipeptide and benzylpenicillin hydrolyses but not methanolysis. The acyl-enzyme derived from both types of substrate is thus shielded from external nucleophiles; the shielding is therefore not an effect, direct or indirect, of the thiazolidinyl group in the penicilloyl-enzyme. The class A beta-lactamase of the PC1 plasmid of S. aureus is distinctly different from the above two representatives of that class, in that it does catalyze methanolysis of depsipeptides (but not of benzylpenicillin). The methanolysis kinetics suggest that deacylation is rate-determining at saturation, a conclusion supported by the demonstration of an intermediate during the hydrolysis of m-[[(phenylacetyl)glycyl]oxy]benzoate, subsequent to leaving-group departure. The beta-lactamases have thus been shown to catalyze the hydrolysis of specific depsipeptides with comparable facility to that demonstrated by D-alanyl-D-alanine carboxypeptidase/transpeptidases. The former enzymes, however, differ in being unable to cleave the analogous peptides.     

N-(phenylacetyl)glycyl-D-aziridine-2-carboxylate, an acyclic amide substrate of beta-lactamases: importance of the shape of the substrate in beta-lactamase evolution

Certain acyclic depsipeptides, but not peptides, are substrates of typical beta-lactamases [Pratt, R.F., & Govardhan, C.P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1302]. This may reflect either the greater chemical reactivity of depsipeptides (and of beta-lactams, the natural substrates) than peptides or the greater ease of distortion of the depsipeptide (ester) than the peptide (amide) group into a penicillin-like conformation. The latter explanation has been shown to be more likely by employment of a novel beta-lactamase substrate. N-(phenylacetyl)glycyl-D-aziridine-2-carboxylate, which combines a high chemical reactivity with a close to tetrahedral amide nitrogen atom. Although this substrate was better (higher kcat/KM) than a comparable depsipeptide for beta-lactamases, it was poorer than the depsipeptide for the Streptomyces R61 D-alanyl-D-alanine peptidase (which catalyzes specific peptide hydrolysis). It therefore seems likely that one vital feature of the putative evolution of a DD-peptidase into a beta-lactamase would have been modification of the active site to, on one hand, accommodate bicyclic beta-lactams and, on the other, exclude productive binding of planar acyclic amides. Certain serine beta-lactamases and the R61 DD-peptidase also catalyze methanolysis and aminolysis by D-phenylalanine of the N-acylaziridine. The latter reaction, the first amide aminolysis shown to be catalyzed by a beta-lactamase, is a very close analogue of the transpeptidase reaction of DD-peptidases. The methanolysis reaction appeared to proceed by way of the same acyl-enzyme intermediate as formed from depsipeptides possessing the same acyl moiety as the aziridine. The kinetics of methanolysis were employed to determine whether acylation or deacylation was rate limiting to the hydrolysis reaction under saturating substrate concentrations. The kinetics of the aminolysis reaction, catalyzed by the Enterobacter cloacae P99 beta-lactamase, showed the characteristics of, and were interpreted in terms of, a sequential mechanism previously deduced for depsipeptides and this enzyme [Pazhanisamy, S., & Pratt, R. F. (1989) Biochemistry 28, 6875-6882]. This mechanism features two separate binding sites, only one of which is productive. Strikingly, the binding of the N-acylaziridine to the nonproductive site was very tight, such that essentially all hydrolysis at substrate concentrations above 0.1Km proceeded via the ternary complex; this could also be true of penicillins.     

Papain-catalysed hydrolysis of some hippuric esters. A new mechanism for papain-catalysed hydrolyses

1. The Michaelis–Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k₀, is 2–3sec.⁻¹ and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k₀, less than 2–3sec.⁻¹. It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme–substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis–Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, σ. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-α-amino acid derivatives.     

Molecular dynamics simulations of the acyl-enzyme and the tetrahedral intermediate in the deacylation step of serine proteases

Despite the availability of many experimental data and some modeling studies, questions remain as to the precise mechanism of the serine proteases. Here we report molecular dynamics simulations on the acyl-enzyme complex and the tetrahedral intermediate during the deacylation step in elastase catalyzed hydrolysis of a simple peptide. The models are based on recent crystallographic data for an acyl-enzyme intermediate at pH 5 and a time-resolved study on the deacylation step. Simulations were carried out on the acyl enzyme complex with His-57 in protonated (as for the pH 5 crystallographic work) and deprotonated forms. In both cases, a water molecule that could provide the nucleophilic hydroxide ion to attack the ester carbonyl was located between the imidazole ring of His-57 and the carbonyl carbon, close to the hydrolytic position assigned in the crystal structure. In the "neutral pH" simulations of the acyl-enzyme complex, the hydrolytic water oxygen was hydrogen bonded to the imidazole ring and the side chain of Arg-61. Alternative stable locations for water in the active site were also observed. Movement of the His-57 side-chain from that observed in the crystal structure allowed more solvent waters to enter the active site, suggesting that an alternative hydrolytic process directly involving two water molecules may be possible. At the acyl-enzyme stage, the ester carbonyl was found to flip easily in and out of the oxyanion hole. In contrast, simulations on the tetrahedral intermediate showed no significant movement of His-57 and the ester carbonyl was constantly located in the oxyanion hole. A comparison between the simulated tetrahedral intermediate and a time-resolved crystallographic structure assigned as predominantly reflecting the tetrahedral intermediate suggests that the experimental structure may not precisely represent an optimal arrangement for catalysis in solution. Movement of loop residues 216-223 and P3 residue, seen both in the tetrahedral simulation and the experimental analysis, could be related to product release. Furthermore, an analysis of the geometric data obtained from the simulations and the pH 5 crystal structure of the acyl-enzyme suggests that since His-57 is protonated, in some aspects, this crystal structure resembles the tetrahedral intermediate.     

Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met→Ala168 mutant penicillin acylases from Kluyvera citrophila

The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters K_m and k_cat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met→Ala168 mutation unstabilizes the first transition-state (E··S^≠) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site.     

Structure elucidation of two differentiation inducing factors (DIF-2 and DIF-3) from the cellular slime mould Dictyostelium discoideum.

Acylation of the aldehyde dehydrogenase.NADH complex by acetic anhydride leads to the production of acetaldehyde and NAD+. By monitoring changes in nucleotide fluorescence, the rate constant for acylation of the active site of the *enzyme.NADH complex was found to be 11 +/- 3 s-1. The rate of acylation by acetic anhydride at the group that binds aldehydes on the oxidative pathway is clearly rapid enough to maintain significant steady-state concentrations of the required active-site-acylated *enzyme.NADH intermediate despite the rapid hydrolysis of this *enzyme.acyl.NADH intermediate (5-10 s-1) [Blackwell, Motion, MacGibbon, Hardman & Buckley (1987) Biochem. J. 242, 803-808]. Hence reversal of the normal oxidative pathway can occur. However, although acylation of the aldehyde dehydrogenase.NADH complex by 4-nitrophenyl acetate also occurs rapidly with a rate constant of 10.9 +/- 0.6 s-1, even under the most extreme trapping conditions only very small amounts of acetaldehyde are detected [Loomes & Kitson (1986) Biochem. J. 235, 617-619]. Furthermore enzyme-catalysed hydrolysis of 4-nitrophenyl acetate is limited by the rate of deacylation of a group on the enzyme (0.4 s-1), which is an order of magnitude less than deacylation of the group at the active site (5-10 s-1). It is concluded that the enzyme-catalysed 4-nitrophenyl ester hydrolysis involves a group on the enzyme that is different from the active-site group that binds aldehydes on the normal oxidative pathway.     

Mechanism of Carboxypeptidase Y Catalyzed Hydrolysis and Aminolysis Reactions

The reaction mechanism of carboxypeptidase Y catalyzed reactions is investigated. Presteady state and steady state kinetic measurements are performed on the hydrolysis and aminolysis of an ester and an amide substrate. It is found that deacylation is the rate determining step in hydrolysis of the ester, pivalic acid 4-nitrophenol and acylation in that of the amide, succinyl-L-alanyl-L-alalyl-L-propyl-L-phenylalanine 4-nitroanilide.

The kinetic effects observed in the presence of a nucleophile, L-valine amide, where aminolysis occurs in parallel to the hydrolysis reaction are analysed in details. The results are described satisfactorily by a reaction scheme which involves the binding of the added nucleophile, (i) to the free enzyme, resulting in a simple competitive effect, and (ii) to the acyl-enzyme with the formation of a complex between the enzyme and the aminolysis product, the dissociation of which is rate determining. That scheme can account for both increases and decreases of kinetic parameter values as a function of the nucleophile concentration. There is no indication of binding of the nucleophile to the enzyme-substrate complex before acylation takes place.     

Crystal structure of wild-type penicillin-binding protein 5 from Escherichia coli: implications for deacylation of the acyl-enzyme complex

Penicillin-binding protein 5 (PBP 5) of Escherichia coli functions as a d-alanine carboxypeptidase (CPase), cleaving d-alanine from the C terminus of cell wall peptides. Like all PBPs, PBP 5 forms a covalent acyl-enzyme complex with beta-lactam antibiotics; however, PBP 5 is distinguished by its high rate of deacylation of the acylenzyme complex (t(1/2) approximately 10 min). A Gly105 --> Asp mutation in PBP 5 markedly impairs deacylation with only minor effects on acylation, and abolishes CPase activity. We have determined the three-dimensional structure of a soluble form of wild-type PBP 5 at 1.85-A resolution and have also refined the structure of the G105D mutant form of PBP 5 to 1.9-A resolution. Comparison of the two structures reveals that the major effect of the mutation is to disorder a loop comprising residues 74-90 that sits atop the SXN motif of the active site. Deletion of the 74-90 loop in wild-type PBP 5 markedly diminished the deacylation rate of penicillin G with a minimal impact on acylation, and abolished CPase activity. These effects were very similar to those observed in the G105D mutant, reinforcing the idea that this mutation causes disordering of the 74-90 loop. Mutation of two consecutive serines within this loop, which hydrogen bond to Ser110 and Asn112 in the SXN motif, had marked effects on CPase activity, but not beta-lactam antibiotic binding or hydrolysis. These data suggest a direct role for the SXN motif in deacylation of the acyl-enzyme complex and imply that the functioning of this motif is modulated by the 74-90 loop.     

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.     

Horse serum butyrylcholinesterase kinetics: a molecular mechanism based on inhibition studies with dansylaminoethyltrimethylammonium

The kinetics of the hydrolysis of butyrylthiocholine by horse serum butyrylcholinesterase (acylcholine acylhydrolase; BuChE; EC 3.1.1.8) exhibit an activation phenomenon at high substrate concentrations. At least two mechanistic models can account for the enzyme kinetics: one assumes the binding of an additional substrate molecule on the acyl-enzyme intermediate, and the other hypothesizes the existence of a peripheral regulatory site for the substrate. (1-Dimethylaminonaphthalene-5-sulfonamidoethyl)-trimethylammonium perchlorate, a potent reversible inhibitor, appears to affect BuChE activity by binding to a peripheral site. The inhibition is of the mixed type at low substrate concentrations and of the competitive type at high substrate concentrations. This is consistent with a peripheral site for the binding of the substrate responsible for the activation phenomenon.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.