Mono- and binuclear Zn2+-beta-lactamase. Role of the conserved cysteine in the catalytic mechanism

When expressed by pathogenic bacteria, Zn2+-beta-lactamases induce resistance to most beta-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn2+-beta-lactamases together with standard antibiotics. For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn2+-beta-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine. It is shown that both the mono-Zn2+ (mononuclear) and di-Zn2+ (binuclear) Zn2+-beta-lactamases are catalytically active but with different kinetic properties. The mono-Zn2+-beta-lactamase requires the conserved cysteine residue for hydrolysis of the beta-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential. Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates. Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme.     

Familial mutations and zinc stoichiometry determine the rate-limiting step of nitrocefin hydrolysis by metallo-beta-lactamase from Bacteroides fragilis

The diverse members of the metallo-beta-lactamase family are a growing clinical threat evolving under considerable selective pressure. The enzyme from Bacillus cereus differs from the Bacteroides fragilis enzyme in sequence, zinc stoichiometry, and mechanism. To chart the evolution of the more reactive B. fragilis enzyme, we have made changes in an active site cysteine residue as well as in zinc content to mimic that which occurs in the B. cereus enzyme. Specifically, by introducing a C104R mutation into the B. fragilis enzyme, binding of two zinc ions is maintained, but the k(cat) value for nitrocefin hydrolysis is decreased from 226 to 14 s(-)(1). Removal of 1 equiv of zinc from this mutant further decreases k(cat) to 4.4 s(-)(1). In both cases, the observed k(cat) closely approximates that found in the di- and monozinc forms of the B. cereus enzyme (12 and 6 s(-)(1), respectively). Pre-steady-state stopped-flow studies using nitrocefin as a substrate indicate that these enzyme forms share a similar mechanism featuring an anionic intermediate but that the rate-limiting step changes from protonation of that species to the C-N bond cleavage leading to the intermediate. Overall, features that contribute 3.7 kcal/mol toward the acceleration of the C-N bond cleavage step have been uncovered although some of the total acceleration is masked in the steady-state by a change in rate-limiting step. These experiments illustrate one step in the evolution of a catalytic mechanism and, in a larger perspective, one step in the evolution of antibiotic resistance mechanisms.     

Irreversible inhibition of the Mycobacterium tuberculosis beta-lactamase by clavulanate

Members of the beta-lactam class of antibiotics, which inhibit the bacterial d,d-transpeptidases involved in cell wall biosynthesis, have never been used systematically in the treatment of Mycobacterium tuberculosis infections because of this organism's resistance to beta-lactams. The critical resistance factor is the constitutive production of a chromosomally encoded, Ambler class A beta-lactamase, BlaC in M. tuberculosis. We show that BlaC is an extended spectrum beta-lactamase (ESBL) with high levels of penicillinase and cephalosporinase activity as well as measurable activity with carbapenems, including imipenem and meropenem. We have characterized the enzyme's inhibition by three FDA-approved beta-lactamase inhibitors: sulbactam, tazobactam, and clavulanate. Sulbactam inhibits the enzyme competitively and reversibly with respect to nitrocefin. Tazobactam inhibits the enzyme in a time-dependent manner, but the activity of the enzyme reappears due to the slow hydrolysis of the covalently acylated enzyme. In contrast, clavulanate reacts with the enzyme quickly to form hydrolytically stable, inactive forms of the enzyme that have been characterized by mass spectrometry. Clavulanate has potential to be used in combination with approved beta-lactam antibiotics to treat multi-drug resistant (MDR) and extremely drug resistant (XDR) strains of M. tuberculosis.     

Catalytic cycle of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus. Solvent viscosity, deuterium isotope effects, and proton inventory studies

The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) is a 28.5 kDa enzyme with three zinc ions in its active site. Although much is known about the roles that various PLCBc active site amino acids play in binding and catalysis, there is little information about the rate-determining step of the PLCBc-catalyzed hydrolysis of phospholipids and the catalytic cycle of the enzyme. To gain insight into these aspects of the hydrolysis, solvent viscosity variation experiments were conducted to determine whether an external step (substrate binding or product release) or an internal step (hydrolysis) is rate-limiting. The data indicate that the PLCBc-catalyzed reaction is unaffected by changes in solvent viscosity. This observation is inconsistent with the notion of substrate binding or product release being rate-determining and supports the hypothesis that a chemical step is rate-limiting. Furthermore, a deuterium isotope effect of 1.9 and a linear proton inventory plot indicate one proton is transferred in the rate-determining step. These data may be used to formulate a comprehensive catalytic cycle that is for the first time based on experimental evidence. In this mechanism, Asp55 of PLCBc activates an active site water molecule for attack on the phosphodiester bond, the hydrolysis of which is rate-limiting. The phosphorylcholine product is the first to leave the active site, followed by diacylglycerol.     

Mechanism and activation for allosteric adenosine 5'-monophosphate nucleosidase. Kinetic alpha-deuterium isotope effects for the enzyme-catalyzed hydrolysis of adenosine 5'-monophosphate and nicotinamide mononucleotide

The kinetic alpha-deuterium isotope effect on Vmax/Km for hydrolysis of NMN catalyzed by AMP nucleosidase at saturating concentrations of the allosteric activator MgATP2- is kH/kD = 1.155 +/- 0.012. This value is close to that reported previously for the nonenzymatic hydrolysis of nucleosides of related structure, suggesting that the full intrinsic isotope effect for enzymatic NMN hydrolysis is expressed under these conditions; that is, bond-changing reactions are largely or completely rate-determining and the transition state has marked oxocarbonium ion character. The kinetic alpha-deuterium isotope effect for this reaction is unchanged when deuterium oxide replaces water as solvent, corroborating this conclusion. Furthermore, this isotope effect is independent of pH over the range 6.95-9.25, for which values of Vmax/Km change by a factor of 90, suggesting that the isotope-sensitive and pH-sensitive steps for AMP-nucleosidase-catalyzed NMN hydrolysis are the same. Values of kH/kD for AMP nucleosidase-catalyzed hydrolysis of NMN decrease with decreasing saturation of enzyme with MgATP2- and reach unity when the enzyme is less than half-saturated with this activator. This requires that the rate-determining step changes from cleavage of the covalent C-N bond to one which is isotope-independent. In contrast to the case for NMN hydrolysis, AMP nucleosidase-catalyzed hydrolysis of AMP at saturating concentrations of MgATP2- shows a kinetic alpha-deuterium isotope effect of unity. Thus, covalent bond-changing reactions are largely or completely rate-determining for hydrolysis of a poor substrate, NMN, but make little or no contribution to rate-determining step for hydrolysis of a good substrate, AMP, by maximally activated enzyme. This behavior has several precedents.     

Dynamics of proton transfer and enzymatic activity

The rate-determining elementary reaction step, i.e. proton transfer from the chymotrypsin active centre to the scissile substrate bond has been studied in the present work. On the basis of our theoretical results a hypothesis was formulated to explain chymotrypsin enzymatic efficiency. After ES complex formation excited vibrational states are populated in the enzyme molecule. In the rate-determining elementary reaction step, the proton transfer takes place from the first excited vibrational state of the N-H bond in the imidazole group of His57. This proton transfer is realised by quantum mechanical tunneling mechanism.     

Delta 2- and delta 3-cephalosporins, penicillinate and 6-unsubstituted penems. Intrinsic reactivity and interaction with beta-lactamases and D-alanyl-D-alanine-cleaving serine peptidases.

The intrinsic reactivity of delta 2- and delta 3-deacetoxy-7-phenylacetamidocephalosporanates, penicillanate, unsubstituted, 2-methyl- and 2-phenyl-penems and other beta-lactam antibiotics has been expressed in terms of the second-order rate constant (M-1.s-1(OH-)) for the hydrolysis of the beta-lactam amide bond by OH- at 37 degrees C. The values thus obtained have been compared with the second-order rate constants (M-1.s-1(enzyme) for the opening of the same beta-lactam amide bond during interaction with the beta-lactamases of Streptomyces albus G and Actinomadura R39 and the D-alanyl-D-alanine-cleaving serine peptidases of Streptomyces R61 and Actinomadura R39. Depending on the cases, the accelerating effect due to enzyme action and expressed by the ratio M-1.s-1(enzyme)/M-1.s-1(OH) varies from less than 2 to more than 1 x 10(6). The primary parameter that governs enzyme action is the goodness of fit of the beta-lactam molecule to the enzyme cavity rather than its intrinsic reactivity. With the D-alanyl-D-alanine-cleaving serine peptidases, the three penems studied form intermediate complexes characterized by very short half lives of 14-100 s, values significantly lower than those exhibited by most beta-lactam compounds.     

Kinetic investigation of soybean trypsin-like enzyme catalysis

Various esters and amides of benzoylarginine and of benzyloxycarbonylarginine were subjected to enzymic hydrolysis at pH 8.5 and 7.2 by soybean trypsin-like enzyme (STLE). The kcat values for the hydrolysis of esters and amides were essentially identical regardless of the kind of leaving group. These results suggest that the STLE-catalyzed hydrolysis of ester and amide substrates proceeds via an acylenzyme intermediate and that the deacylation step is rate-determining. Hydrolysis of various 4-methylcoumaryl-7-amides of varying chain length and amino acid sequence was carried out at pH 8.5. Analysis of kinetic parameters revealed that STLE does not exhibit any remarkable subsite requirement, but somewhat preferentially hydrolyzes shorter substrates. These observations are consistent with the fact that STLE does not hydrolyze protein substrates or oxidized insulin B chain but hydrolyzes oligopeptides (Nishikata, M. (1984) J. Biochem. 95, 1169-1177). It is possible that the active site of STLE is located at a deep position in the enzyme molecule. From the pH dependency of kcat/Km, the participation of a histidine residue in the catalytic process of STLE was suggested.     

Mechanistic requirements for the efficient enzyme-catalyzed hydrolysis of thiosialosides

The sialidase from Micromonospora viridifaciens has been found to catalyze the hydrolysis of aryl 2-thio-alpha-D-sialosides with remarkable efficiency: the first- and second-order rate constants, kcat and kcat/Km, for the enzyme-catalyzed hydrolysis of PNP-S-NeuAc are 196 +/- 5 s(-1) and (6.7 +/- 0.7) x 10(5) M(-1) s(-1), respectively. A reagent panel of eight aryl 2-thio-alpha-D-sialosides was synthesized and used to probe the mechanism for the M. viridifaciens sialidase-catalyzed hydrolysis reaction. In the case of the wild-type enzyme, the derived Br?nsted parameters (beta(lg)) on kcat and kcat/Km are -0.83 +/- 0.11 and -1.27 +/- 0.17 for substrates with thiophenoxide leaving groups of pKa values > or = 4.5. For the general-acid mutant, D92G, the derived beta(lg) value on kcat for the same set of leaving groups is -0.82 +/- 0.12. When the conjugate acid of the departing thiophenol was < or = 4.5, the derived Br?nsted slopes for both the wild-type and the D92G mutant sialidase were close to zero. In contrast, the nucleophilic mutant, Y370G, did not display a similar break in the Br?nsted plots, and the corresponding values for beta(lg), for the three most reactive aryl 2-thiosialosides, on kcat and kcat/Km are -0.76 +/- 0.28 and -0.84 +/- 0.04, respectively. Thus, for the Y370G enzyme glycosidic C-S bond cleavage is rate-determining for both kcat and kcat/Km, whereas, for both the wild-type and D92G mutant enzymes, the presented data are consistent with a change in rate-determining step from glycosidic C-S bond cleavage for substrates in which the pKa of the conjugate acid of the leaving group is > or = 4.5, to either deglycosylation (kcat) or a conformational change that occurs prior to C-S bond cleavage (kcat/Km) for the most activated leaving groups. Thus, the enzyme-catalyzed hydrolysis of 2-thiosialosides is strongly catalyzed by the nucleophilic tyrosine residue, yet the C-S bond cleavage does not require the conserved aspartic acid residue (D92) to act as a general-acid catalyst.     

Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis

The L1 metallo-beta-lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in beta-lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 beta-lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of beta-lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K(m) values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K(m) values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for beta-lactam hydrolysis, it is clearly important to its activity and substrate specificity.     

Uncoupling the enzymatic and autoprocessing activities of Helicobacter pylori gamma-glutamyltranspeptidase

Gamma-glutamyltranspeptidase (gammaGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the gamma-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported gammaGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the gamma-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian gammaGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the gamma-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions.     

Direct measurement of metal-ion chelation in the active site of the AAA+ ATPase magnesium chelatase

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis. This complex enzyme has at least three substrates and couples ATP hydrolysis to the insertion of Mg2+ into protoporphyrin IX. We directly observed metal-ion chelation fluorometrically, providing the first data describing the on-enzyme reaction. We describe the transient-state kinetics of magnesium chelatase with direct observation of the evolution of an enzyme-product complex EMgDIX. We demonstrate that MgATP2- binding occurs after the rate-determining step. As nucleotide hydrolysis is essential for the overall reaction this must also occur after the rate-determining step. This provides the first evidence for the synchronization of the ATPase and chelatase pathways and suggests a mechanism where nucleotide binding acts to clamp the chelatase in a product complex. Comparison of rate constants for the slow step in the reaction with further transient kinetics under conditions where multiple turnovers can occur reveals that an additional activation step is required to explain the behavior of magnesium chelatase. These data provide a new view of the sequence of events occurring in the reaction catalyzed by magnesium chelatase.     

Kinetic mechanism of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with conversion of NAD+ to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. IMPDH is a target for antitumor, antiviral, and immunosuppressive chemotherapy. We have determined the complete kinetic mechanism for IMPDH from Tritrichomonas foetus using ligand binding, isotope effect, pre-steady-state kinetic, and rapid quench kinetic experiments. Both substrates bind to the free enzyme, which suggests a random mechanism. IMP binds to the enzyme in two steps. Two steps are also involved when IMP binds to a mutant IMPDH in which the active site Cys is substituted with a Ser. This observation suggests that this second step may be a conformational change of the enzyme. No Vm isotope effect is observed when [2-2H]IMP is the substrate which indicates that hydride transfer is not rate-limiting. This result is confirmed by the observation of a pre-steady-state burst of NADH production when monitored by absorbance. However, when NADH production was monitored by fluorescence, the rate constant for the exponential phase is 5-10-fold lower than when measured by absorbance. This observation suggests that the fluorescence of enzyme-bound NADH is quenched and that this transient represents NADH release from the enzyme. The time-dependent formation and decay of [14C]E-XMP intermediates was monitored using rapid quench kinetics. These experiments indicate that both NADH release and E-XMP hydrolysis are rate-limiting and suggest that NADH release precedes hydrolysis of E-XMP.     

Interaction of beta-iodopenicillanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39.

The beta-lactamases of Streptomyces albus G and Actinomadura R39 are inactivated by beta-iodopenicillanate. However, in contrast with the beta-lactamase I from Bacillus cereus, they also efficiently catalyse the hydrolysis of the inactivator; with the S. albus G enzyme, kcat. is larger than 25s-1 and the number of turnovers before inactivation is 515. With the A. R39 enzyme, kcat. is larger than 50s-1 and the number of turnovers before inactivation is 80. After hydrolysis of the beta-lactam amide bond, the product rearranges into 2.3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which exhibits an absorption maximum at 305 nm.     

Histidine ammonia-lyase. The use of 4-fluorohistidine in identification of the rate-determining step.

The alpha,beta eliminations of NH3 from L-histidine and 4-fluoro-L-histidine by histidine ammonia-lyase appear to occur by similar mechanisms, although a large difference in Vmax for the two reactions was observed. Both reactions were shown to be reversible with an equilibrium constant of 4 to 5. The presteady state kinetics of the deamination of 4-fluoro-L-histidine indicates that the rate-determining step precedes the dissociation of ammonia from the enzyme. The isotope effect of 1.4 to 2.0 observed with 4-fluoro-DL-[beta-2-H2]histidine or DL-[beta-2-H2]histidine or DL-[beta-2-H2]histidine indicates that the C-H bond breakage is at least partially rate-determining for the deamination of both substrates.     

Kinetic analysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides

The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic "burst". This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes any other residue present in the gorge for being the catalytic nucleophile pole.     

Zinc and antibiotic resistance: metallo-β-lactamases and their synthetic analogues

Antibiotic resistance to clinically employed beta-lactam antibiotics currently poses a very serious threat to the clinical community. The origin of this resistance is the expression of several beta-lactamases that effectively hydrolyze the amide bond in beta-lactam compounds. These beta-lactamases are classified into two major categories: serine beta-lactamases and metallo-beta-lactamases. The metalloenzymes use one or two zinc ions in their active sites to catalyze the hydrolysis of all classes of beta-lactam antibiotics, including carbapenems. As there is no clinically useful inhibitor for the metallo-beta-lactamases, it is important to understand the mechanism by which these enzymes catalyze the hydrolysis of antibiotics. In this regard, the development of synthetic analogues will be very useful in understanding the mechanism of action of metallo-beta-lactamases. This review highlights some important contributions made by various research groups in the area of synthetic analogues of metallo-beta-lactamases, with major emphasis on the role of dinuclear Zn(II) complexes in the hydrolysis of beta-lactam antibiotics.     

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.     

Isomer-specific proteolysis of model substrates: influence that the location of the proline residue exerts on cis/trans specificity

In an effort to further develop the technique of isomer-specific proteolysis, a number of proline-containing substrates were subjected to hydrolysis in the presence of chymotrypsin, trypsin, or prolidase. The objective was to determine whether direct hydrolysis of the cis form of the substrate could occur and, if so, the extent to which it is slower than the hydrolysis of the equivalent trans form. It is shown that for both peptide and amide substrates, which contain proline at the P2 position, the cis form can be hydrolyzed directly by either chymotrypsin or trypsin, in contrast to earlier suggestions in the literature. For similar amide substrates, it was found that chymotrypsin has a lower catalytic efficiency for the cis form, relative to the trans form, by a factor of 20 000 while, for trypsin and its substrate, the cis form was cleaved about 2000 times less efficiently. Results for a trypsin substrate with proline at the P2' position, rather than the P2 position, were quite different however, since there was no indication that the cis form could be directly cleaved even at the highest enzyme concentration. There was also no indication that prolidase could cleave the dipeptide Phe-Pro when the active bond itself is in the cis form. These collective results suggest that the ability of proteases to cleave a substrate with a cis peptide bond depends strongly on the location of the cis bond relative to the active bond that is being cleaved.     

Elucidation of the chemical nature of the steady-state intermediates in the mechanism of carboxypeptidase A

Cryospectrokinetic studies of zinc and cobalt carboxypeptidase A disclosed two intermediates in the hydrolysis of both peptides and depsipeptides and furnished all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2---E + P [Auld, D. S., Galdes, A., Geoghegan, K. F., Holmquist, B., Martinelli, R. A., & Vallee, B. L. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5041-5045]. Since the ES2 intermediate is the predominate enzyme species present at steady state, its chemical nature is deducible from subzero chemical quench studies done after steady state is established. Extrapolation of the product concentration to zero time, [P0], measures the concentration of the enzyme species in which bond cleavage has occurred. For peptides, the [P0]values are zero, indicating that no product is generated prior to turnover and therefore the ES2 intermediate involves a complex between enzyme and intact peptide substrate. For depsipeptides, [P0] values are 1 mol of produce per mole of enzyme over the entire temperature range -20 to -50 degrees C, indicating cleavage of the ester bond occurs prior to the rate-limiting step so that ES2 is more properly denoted by EP1P2, where P1 and P2 are the substrates for the reverse reaction. The rate-limiting step for depsipeptides thus involves release of the products which may occur directly or through a mandatory conformational change followed by rapid product release.