Stephen G. Waley

Automated analysis of enzyme inactivation phenomena. Application to beta-lactamases and DD-peptidases.

In the presence of a reporter substrate, the progressive inactivation of an enzyme was easily studied by directly transmitting absorbance readings to a microcomputer. Pseudo-first order rate constants as high as 0.3 sec-1 were rapidly and accurately measured. When utilization of the reporter substrate did not exceed 10%, the rate of the reaction (vt) could be considered as proportional to the active enzyme concentration at any time during the analysis and the decrease of vt was first order with time. This simple method was used to follow the inactivation of beta-lactamases (EC 3.5.2.6) by various physical and chemical agents. When a large proportion (30-80%) of reporter substrate was destroyed, a correction was introduced to account for the corresponding decrease of its rate of utilization. This enabled experiments to be performed with a DD-peptidase and a substrate exhibiting a low delta epsilon upon hydrolysis. For the first time, the inactivation of a penicillin-sensitive enzyme by a beta-lactam could be continuously and directly observed. Finally, the method was extended to the study of hysteresis phenomena.

Penicillinase active sites: Labelling of serine-44 in β-lactamase I by 6β-bromopenicillanic acid

fi-Lactamases inactivate penicillins and cephalosporins by catalysing the hydrolysis of the P-lactamase ring in these ~lini~~ly-v~uable antibiotics. The resistance of pathogenic bacteria is frequently due to their production of p-lactamases. Yet we know little in chemical terms about catalysis by these enzymes, Until now, affinity labelling has not been carried to a successful conclusion with any fl-lactamase. @-Lactamase I from ~~~~~u~ cereus has been used here ; it is readily prepared [l], the sequence has been studied [2], it is homologous with the enzymes from pathogens whose structure is known [3], and it is being examined crystallographically [4f. The reagent used here for affinity labelling, 6~.bromopeni~ill~ic acid, inactivates &lactamase I in a rapid, stoicheiometric reaction IS], and the rate of inactivation is decreased by the presence of substrate [6]. We now report that the site labelled is serine44. This result invites us to regard certain /3-lactamases as serine enzymes.

Use of electrospray mass spectrometry to directly observe an acyl enzyme intermediate in β-lactamase catalysis

Electrospray mass spectrometry was used to directly observe intact acyl enzyme complexes formed between a class C β‐lactamase (from Enterobacter cloacae P99) and four poor substrates/inhibitors. In each case the molecular weight difference between the unreacted and the reacted β‐lactamase was consistent with the formation of an acyl enzyme.

The form of 2-phosphoglycollic acid bound by triosephosphate isomerase

Abstract In NMR experiments designed to distinguish between possible enzyme-bound forms of the inhibitor 2-phosphoglycollic acid, it is found that neither of the di-anionic species, that would be consistent with the observed pH-dependence of K i , is in fact correct. Instead, the enzyme appears to bind the tri-anionic species of this inhibitor, taking up a proton at a separate site.

The amino acid sequence of the zinc-requiring β-lactamase II from the bacterium Bacillus cereus 569

The amino acid sequence of the zinc‐requiring β‐Mactamase II from Bacillus cereus strain 569 has been determined. It consists of a single polypeptide chain of 227 residues. It is the only example so far fully characterized of a class B β‐lactamase, and is structurally and mechanistically distinct from both the widely distributed class A β‐lactamases (such as the Escherichia coli RTEM enzyme) and from the chromosomally encoded class C enzymes from Gram‐negative bacteria.

Direct observation of a tetrahedral boronic acid–β-lactamase complex using 11B NMR spectroscopy

A tetrahedral boronic acid–β-lactamase complex formed by treatment of the P99 β-lactamase enzyme from Enterobacter cloacae with 3-dansylamidophenylboronic acid has been directly observed using 11B NMR spectroscopy.

On the chemical binding of 6β-bromopenicillanic acid to β-lactamase I

6β-Bromopenicillanic acid (1) binds to serine-44 of the β-lactamase I from B. cereus as a dihydrothiazine derivative, which undergoes further chemical changes during cleavage of the enzyme.

Use of electrospray mass spectrometry to investigate the inhibition of β-lactamases by 6-halogenopenicillanic acids

Electrospray mass spectrometry has been used to investigate the mechanism of inhibition of class A and C β-lactamases by 6-halogenopenicillanic acids; in the case of the 6β-halogen substituted penicillanic acids the molecular mass differences observed between the unreacted and the inhibited β-lactamases are consistent with the formation of an enzyme bound dihydrothiazine derivative, as previously proposed.

β-lactamase action: Isolation of an active-site serine peptide from the Pseudomonas enzyme and a penicillin

j?-Lactamases (penicillinases) comprise an efficient and diverse group of enzymes that are often responsible for the resistance of pathogens to &lactam antibiotics. An inducible, cell-bound chromosomal P-lactamase from Pseudomonas aeruginosa NCTC 8203 was first described by Sabath et al. [ 1 ] and was later obtained in partially purified form [2]. A mutant of Ps. aeruginosa 18s was found to produce a similar /3-lactamase constitutively [3], and this enzyme has been purified to apparent homogeneity [4]. Those P-lactamases that have been studied structurally fall into two classes, A and B [5]. The Pseudomonas enzyme however, with its relatively highMr [2,4], has been put forward as a candidate for a third structural class [5]. We suggested in [6] that class A fl-lactamases might be serine enzymes, serine being the amino acid residue labelled by a specific inactivator. This hypothesis is now broadened: we identify a penicilloyl-serine at the active site of the Pseudomonas enzyme, by the isolation of a labile acyl-enzyme.

On the chemistry of β-lactamase inhibition by 6β-bromopenicillanic acid

6β-Bromopenicillanic acid, a powerful inhibitor of β-lactamase I from Bacillus cereus, reacts with a serine residue in the enzyme and is bound, via an ester linkage, as the dihydrothiazine (2a). Spectroscopic and chemical evidence is presented for this assignment and the evidence compared to that obtained from the related model dihydrothiazine (2b). Under certain conditions the bound species underwent further chemical changes caused by an autoxidation reaction; the model dihydrothiazine (2b) undergoes similar autoxidation reactions.