A suicide-substrate mechanism for hydrolysis of beta-lactams by an anti-idiotypic catalytic antibody

The catalytic mechanism of an anti-idiotypic antibody, 9G4H9, displaying a beta-lactamase activity was investigated. Kinetics experiments suggest that some penicillinic derivatives behave both as substrates and inactivators. Biochemical and immunological experiments strongly indicate that ampicillin may be regarded as a suicide substrate for hydrolysis by 9G4H9. The anti-idiotypic network appears as a way to create enzyme mimics with modified catalytic activities.     

Interactions between non-classical beta-lactam compounds and the beta-lactamases of Actinomadura R39 and Streptomyces albus G.

6-Aminopenicillanic acid, 7-aminocephalosporanic acid, mecillinam and quinacillin have varying substrate activities for both the R39 beta-lactamase (excreted by Actinomadura R39) and the G beta-lactamase (excreted by Streptomyces albus G). Cefoxitin and quinacillin sulphone are not recognized by the G beta-lactamase and are weak inactivators of the R39 beta-lactamase. N-Formimidoylthienamycin is a poor substrate for the G beta-lactamase and a potent inactivator of the R39 beta-lactamase. The high value of the bimolecular rate constant for enzyme inactivation is mainly due to a very low dissociation constant (1 microM). Clavulanate is an inactivator of both G and R39 beta-lactamases. The reaction with this latter enzyme is a branched pathway where normal turnover and permanent enzyme inactivation occur concomitantly. Between 28 and 43 molecules of clavulanate are hydrolysed before one of them has the opportunity to inactivate one molecule of enzyme.     

Evaluation of inhibition of the carbenicillin-hydrolyzing beta-lactamase PSE-4 by the clinically used mechanism-based inhibitors

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.     

The active site of the P99 beta-lactamase from Enterobacter cloacae.

Labelling the beta-lactamase of Enterobacter cloacae P99 with a poor substrate or a mechanism-based inactivator points to an active-site serine residue in a sequence closely resembling that of the ampC beta-lactamase. These results establish the P99 enzyme as a class-C beta-lactamase, and the concurrence of the two approaches helps to confirm the reliability of determining active-site sequences with the aid of mechanism-based inactivators.     

X-ray structure of the Asn276Asp variant of the Escherichia coli TEM-1 beta-lactamase: direct observation of electrostatic modulation in resistance to inactivation by clavulanic acid.

The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules. Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution. The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant. Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences. The report herein represents the best studied example of inhibitor-resistant beta-lactamases.     

6-(Methoxymethylene)penicillanic acid: inactivator of RTEM beta-lactamase from Escherichia coli

The Z and E isomers of 6-(methoxymethylene)-penicillanic acid have been synthesized, and their interaction with the RTEM beta-lactamase has been studied. The Z isomer is an inhibitor and an inactivator of the enzyme, and there is some similarity between its behavior and that of other mechanism-based inactivators such as clavulanic acid and the penam sulfones. Kinetic analysis of the interaction of the enzyme with the Z isomer has allowed a detailed evaluation of the factors that are important in the design of anti-beta-lactamase agents. In contrast to the Z compound, the E isomer of 6-(methoxymethylene)penicillanic acid is not a substrate, an inhibitor, or an inactivator of the enzyme.     

Inhibition of bovine plasma amine oxidase by 1,4-diamino-2-butenes and -2-butynes

Bovine plasma amine oxidase (BPAO) was previously shown to be irreversibly inhibited by propargylamine and 2-chloroallylamine. 1,4-Diamine versions of these two compounds are here shown to be highly potent inactivators, with IC50 values near 20 microM. Mono-N-alkylation or N,N-dialkylation greatly lowered the inactivation potency in every case, whereas the mono-N-acyl derivatives were also weaker inhibitors and enzyme activity was recoverable. The finding that the bis-primary amines 1,4-diamino-2-butyne (a known potent inhibitor of diamine oxidases) and Z-2-chloro-1,4-diamino-2-butene are potent inactivators of BPAO is suggestive of unexpected similarities between plasma amine oxidase and the diamine oxidases and implies that it may be unwise to attempt to develop selective inhibitors of diamine oxidase using a diamine construct.     

Beta-lactamase TEM1 of E. coli. Crystal structure determination at 2.5 A resolution.

The crystal structure of beta-lactamase TEM1 from E. coli has been solved to 2.5 A resolution by X-ray diffraction methods and refined to a crystallographic R-factor of 22.7%. The structure was determined by multiple isomorphous replacement using four heavy atom derivatives. The solution from molecular replacement, using a polyalanine model constructed from the C alpha coordinates of S. Aureus PCl enzyme, provided a set of phases used for heavy atom derivatives analysis. The E. coli beta-lactamase TEM1 is made up of two domains whose topology is similar to that of the PCl enzyme. However, global superposition of the two proteins shows significant differences.     

The use of neamine as a molecular template: Inactivation of bacterial antibiotic resistance enzyme aminoglycoside 3′-phosphotransferase type IIa

Aminoglycoside 3′-phosphotransferase type IIa [APH(3′)-IIa] is a member of the family of bacterial aminoglycoside-modifying enzymes. Bacteria that harbor these enzymes are resistant to aminoglycoside antibiotics. Four aminoglycoside-based affinity inactivators were synthesized and were shown to be both substrates and inactivators for APH(3′)-IIa. These affinity inactivators are N-bromoacetylated derivatives of neamine, an aminoglycoside antibiotic, where the bromoacetyl moiety in each was introduced regiospecifically at a different amine of the parent compound.     

The relative catalytic efficiency of beta-lactamase catalyzed acyl and phosphyl transfer

Phosphonamidates which bear a simple resemblance to penicillin type structures have been synthesised as potential inhibitors of beta-lactamases: -ethyl N-(benzyloxycarbonyl) amidomethyl phosphonyl amides, PhCH(2)OCONHCH(2)P(O)(OEt)NR(2), the amines HNR(2) being l-proline, d-proline, l-thiazolidine, and o-anthranilic acid. The proline derivatives completely and irreversibly inactivated the class C beta-lactamase from Enterobacter cloacae P99, in a time-dependent manner, indicative of covalent inhibition. The inactivation was found to be exclusive to the class C enzyme and no significant inhibition was observed with any other class of beta-lactamase. The anthranilic acid derivative exhibited no appreciable inactivation of the beta-lactamases. The phosphonyl proline and phosphonyl thioproline derivatives were separated into their diastereoisomers and their individual second order rate constants for inhibition were found to be 7.72 +/- 0.37 and 8.3 x 10(-2) +/- 0.004 M(-1) s(-1) for the l-proline derivatives, at pH 7.0. The products of the inhibition reaction of each individual diastereoisomer, analyzed by electrospray mass spectroscopy, indicate that the more reactive diastereoisomers phosphonylate the enzyme by P-N bond fission with the elimination of proline. Conversely, gas chromatographic detection of ethanol release by the less reactive proline diastereoisomer suggests phosphonylation occurs by P-O bond fission. The enzyme enhances the rate of phosphonylation with P-N fission by at least 10(6) compared with that effected by hydroxide-ion. The pH dependence of the rate of inhibition of the beta-lactamase by the more reactive diasteroisomer is consistent with the reaction of the diprotonated form of the enzyme, EH(2), with the inhibitor, I (or its kinetic equivalents EH with IH). This pH dependence and the rate enhancement indicate that the enzyme appears to use the same catalytic apparatus for phosphonylation as that used for hydrolysis of beta-lactams. The stereochemical consequences of nucleophilic displacement at the phosphonyl centre are discussed.     

Cephalosporinase and penicillinase activities of a β-lactamase from Pseudomonas pyocyanea

1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.     

Conjugated alpha-keto acids as mechanism-based inactivators of brewer's yeast pyruvate decarboxylase: electronic effects of substituents and detection of a long-lived intermediate

A series of phenyl substituted E-4-phenyl-2-keto-3-butenoic acid derivatives were synthesized (p-Cl, m-Cl, p-NO2, m-NO2, o-NO2, 3,4-Cl2, 2,6-Cl2, p-CH3O, p-(CH3)2N) and tested as potential irreversible inhibitors of brewer's yeast pyruvate decarboxylase (EC 4.1.1.1). All those derivatives with electron withdrawing substituents were found to be time-dependent inactivators of the enzyme, unlike the p-CH3O- and p-(CH3)2N derivatives. Detailed kinetic studies with the m-nitro derivative (the most potent inhibitor) indicated that this compound formed reversible complexes with the enzyme at two sites (supposed regulatory and catalytic with Ki values of 0.026 and 0.13 mM, respectively) prior to irreversible inactivation of the enzyme. In addition, concurrently with the inactivation, addition of the m-NO2 derivative to the enzyme produced a new VIS absorbance with lambda max near 430 nm. This absorbance was attributed to the enzyme-bound enamine intermediate. The time course of formation and disappearance of the intermediate could be determined and provided detailed information about the mechanism of the enzyme.     

Active site directed inactivators of mouse submaxillary renin.

The following active site directed inactivators for the pressor enzyme renin were synthesized: L-alpha-bromo-isocaproyl(BIC)-Leu-Val-Tyr-Ser-OH, L-BIC-Val-Tyr-Ser-OH, L-BIC-Leu-Val-OCH3, L-BIC-Leu-Val-OH, L-BIC-Val-Tyr-NH2, L-BIC-Val-Tyr-OCH3, L-BIC-Val-Tyr-OH, L-BIC-Leu-OCH3, L-BIC-Val-OCH3, and L-BIC-OCH3. The rate of inactivation of mouse submaxillary gland renin by these reagents was studied under a variety of conditions. L-alpha-Bromoisocaproyl-Val-Tyr-Ser-OH and L-alpha-bromoisocaproyl-Leu-Val-Tyr-Ser-OH and L-alpha-bromoisocaproyl-Leu-Val-Tyr-Ser-OH were the most efficient inactivators followed by L-alpha-bromoisocaproyl-Val-Tyr-NH2. The rates of inactivation by the first two peptides were strongly dependent on pH, being most efficient at low pH, least efficient at pH near 5.6, and becoming efficient again at neutral pH. The rate of the inactivation by L-alpha-bromoisocaproyl-Val-Tyr-NH2, in which the C-terminal carboxyl group is blocked, was only slightly dependent on pH. Complete inactivation was achieved by these three reagents. The inactivation was accompanied by incorporation of a stoichiometric quantity of the radiolabeled reagents. Based on these findings it was concluded that the inactivators reacted with a carboxyl group(s) in the active site of the renin molecule to form an esteric linkage. These data also suggest that a carcoxyl group(s) may constitute part of the catalytically essential functional groups in renin action. D-alpha-Bromoisocaproyl derivatives of the various peptides mentioned above were also prepared. These compounds were much less active than the L isomers indicating that the inactivation by the L-alpha-bromoisocaproyl peptides was a specific reaction.     

Probing the mechanism of a membrane transport protein with affinity inactivators

Affinity inactivators are useful for probing catalytic mechanisms. Here we describe the synthesis and properties of methanethiosulfonyl (MTS) galactose or glucose derivatives with respect to a well studied membrane transport protein, the lactose permease of Escherichia coli. The MTS-galactose derivatives behave as affinity inactivators of a functional mutant with Ala(122)-->Cys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (Deltamu(H(+))) markedly increases the rate of reaction between Cys(122) and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala(122)-->Cys mutation is combined with a mutation (Cys(154)-->Gly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and Deltamu(H(+)) has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala(122) in helix IV. In addition, the findings demonstrate that the MTS-galactose derivatives do not react with the Cys residue at position 122 upon binding per se but at a subsequent step in the overall transport mechanism. Thus, these inactivators behave as unique suicide substrates.     

Using the beta-lactamase protein-fragment complementation assay to probe dynamic protein-protein interactions

We have developed a general experimental strategy that enables the quantitative detection of dynamic protein-protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. We have described a number of assays with different reporter readouts, but of particular value to studies of protein interaction dynamics are assays based on enzyme reporters that catalyze the creation of products, thus taking advantage of the amplification of signal afforded. Here we describe protocols for one such PCA based on the enzyme TEM beta-lactamase as a reporter in mammalian cells. The beta-lactamase PCA consists of fusing complementary fragments of beta-lactamase to two proteins of interest. If the proteins interact, the fragments are brought together and fold into active beta-lactamase. Here we describe a protocol for this PCA that can be completed in a few hours, using two different substrates that are converted to fluorescent or colored products by beta-lactamase.     

Clues to the development of mechanism-based inactivators of 3 alpha-hydroxysteroid dehydrogenase: comparison of steroidal and nonsteroidal Michael acceptors and epoxides.

A series of steroidal and nonsteroidal Michael acceptors that represent reaction products for 3 alpha-hydroxysteroid dehydrogenase were synthesized and evaluated as potential enzyme-generated inactivators. Introduction of exocyclic olefins either at C-2 or C-6 produced inhibitors with high affinity for the enzyme (0.05 to 5.0 microM). However, despite this affinity, none of these compounds produced time-dependent inactivation of the enzyme. By contrast, analogs based on 1-phenyl-2-propen-1-one were stoichiometric inactivators of the enzyme and ease of turnover of the parent latent Michael acceptor depended on the presence of an electron-withdrawing substituent at the para position. A series of steroidal and nonsteroidal epoxides in which the oxiranyl oxygen could be substituted for the 3-ketone (the acceptor carbonyl of a steroid substrate) were also synthesized and evaluated as potential mechanism-based inactivators. Steroidal 2 alpha,3 alpha-, and 3 alpha,4 alpha-epoxides as well as 3 alpha- and 3 beta-spiroepoxides did not bind to the enzyme and were unable to cause enzyme inactivation in either the presence or absence of pyridine nucleotide. In contrast, nitrostyrene oxides produced time-dependent inactivation, the rate of which was governed by the presence of an electron withdrawing group at the para position. These data indicate that the design of mechanism-based inactivators for 3 alpha-hydroxysteroid dehydrogenase requires the incorporation of electron-withdrawing groups adjacent to the latent enzyme-activated group and, as a result, the turnover and/or reactivity of these compounds is increased. Moreover, these compounds can be modeled on nonsteroids.     

Structural and kinetic analysis of two covalent sialosyl-enzyme intermediates on Trypanosoma rangeli sialidase

Trypanosoma rangeli sialidase is a glycoside hydrolase (family GH33) that catalyzes the cleavage of alpha-2-->3-linked sialic acid residues from sialoglycoconjugates with overall retention of anomeric configuration. Retaining glycosidases usually operate through a ping-pong mechanism, wherein a covalent intermediate is formed between the carbohydrate and an active site carboxylic acid of the enzyme. Sialidases, instead, appear to use a tyrosine as the catalytic nucleophile, leaving the possibility of an essentially different catalytic mechanism. Indeed, a direct nucleophilic role for a tyrosine was shown for the homologous trans-sialidase from Trypanosoma cruzi, although itself not a typical sialidase. Here we present the three-dimensional structures of the covalent glycosyl-enzyme complexes formed by the T. rangeli sialidase with two different mechanism-based inactivators at 1.9 and 1.7 Angstroms resolution. To our knowledge, these are the first reported structures of enzymatically competent covalent intermediates for a strictly hydrolytic sialidase. Kinetic analyses have been carried out on the formation and turnover of both intermediates, showing that structural modifications to these inactivators can be used to modify the lifetimes of covalent intermediates. These results provide further evidence that all sialidases likely operate through a similar mechanism involving the transient formation of a covalently sialylated enzyme. Furthermore, we believe that the ability to "tune" the inactivation and reactivation rates of mechanism-based inactivators toward specific enzymes represents an important step toward developing this class of inactivators into therapeutically useful compounds.     

Inactivation of the Pseudomonas striata broad specificity amino acid racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide, and mechanistic studies

Mechanism-based inactivators were used to probe the active site of the broad specificity amino acid racemase from Pseudomonas striata. Kinetic parameters for the inactivation of the racemase with both stereoisomers of beta-fluoroalanine, beta-chloroalanine, and O-acetylserine were determined. By use of 14C-labeled O-acetylserines, the stoichiometry of inactivator binding was found to be one inactivator bound per enzyme subunit. The PLP-dependent enzyme contains one coenzyme per subunit, and after NaB3H4 reduction of the PLP-imine bond, followed by trypsin digestion of the protein, the amino acid sequence of the PLP-binding peptide was determined. Trypsin digestion of the enzyme labeled with either L or D isomer of O-acetylserine and sequencing of the labeled peptide revealed that the inactivators bind to the same lysine residue which binds PLP in native enzyme. The characterization of a PLP adduct released from inactivated enzyme under some conditions is also described. Implications of the formation of this compound with respect to the overall reaction mechanism of inactivation are discussed.     

Overcoming resistance to beta-lactamase inhibitors: comparing sulbactam to novel inhibitors against clavulanate resistant SHV enzymes with substitutions at Ambler position 244

Amino acid changes at Ambler position R244 in class A TEM and SHV beta-lactamases confer resistance to ampicillin/clavulanate, a beta-lactam/beta-lactamase inhibitor combination used to treat serious infections. To gain a deeper understanding of this resistance phenotype, we investigated the activities of sulbactam and two novel penem beta-lactamase inhibitors with sp2 hybridized C3 carboxylates and bicyclic R1 side chains against a library of SHV beta-lactamase variants at the 244 position. Compared to SHV-1 expressed in Escherichia coli, all 19 R244 variants exhibited increased susceptibility to ampicillin/sulbactam, an important difference compared to ampicillin/clavulanate. Kinetic analyses of SHV-1 and three SHV R244 (-S, -Q, and -L) variants revealed the Ki for sulbactam was significantly elevated for the R244 variants, but the partition ratios, kcat/kinact, were markedly reduced (13 000 --> 相似文献