Biomedical subjects

S G Waley

Publications and source records attributed to S G Waley.

At least 37 records · Page 2Linked to original sources

PubMed1987

beta-lactamase I from Bacillus cereus. Structure and site-directed mutagenesis.

The sequence of the gene for beta-lactamase I from Bacillus cereus 569/H has been redetermined. Oligonucleotide-directed mutagenesis has been carried out, and the effects of the changes on the ampicillin-resistance of Escherichia coli TG1 expressing the mutant genes have been studied. Lysine-73, close to the active-site serine-70 and a highly-conserved residue, has been converted into arginine. This change had a large effect on activity, but did not abolish it. An even larger effect was found in the mutant in which glutamate-166 had been converted into glutamine; this had little or no activity. On the other hand, the conversion of glutamate-168 into aspartate gave fully active enzyme. Glutamate-166 is an invariant residue, but glutamate-168 is not. Alanine-123 has been replaced by cysteine, to give active enzyme; this change forms part of the plan to introduce a disulphide bond into the enzyme.

Ampicillin Resistance↗

PubMed1987

An X-ray-crystallographic study of beta-lactamase II from Bacillus cereus at 0.35 nm resolution.

Crystals of beta-lactamase II (EC 3.5.2.6., 'penicillinase') from Bacillus cereus were grown with Cd(II) in place of the natural Zn(II) cofactor and stabilized by cross-linking with glutaraldehyde. Their space group is C2, the cell dimensions are a = 5.44 nm, b = 6.38 nm, c = 7.09 nm and beta = 93.6 degrees, and there is one molecule in the asymmetric unit. Diffraction data were collected from cross-linked crystals of the Cd(II)-enzyme, the apoenzyme and six heavy-atom derivatives. The electron-density map calculated at 0.35 nm resolution reveals the essential Cd(II) ion surrounded by three histidine residues and one cysteine residue. The position of a glutamic acid residue, modification of which destroys activity [Little, Emanuel, Gagnon & Waley (1986) Biochem. J. 233, 465-469], suggests the probable location of the active site of the enzyme. Two minor Cd(II) sites not essential for activity were also located. The structure of the apoenzyme at this resolution appears to differ from that of the Cd(II)-enzyme only in the orientation of two of the histidine residues and the cysteine residue that surround the metal ion.

Apoenzymes↗

PubMed1987

Cryoenzymology of beta-lactamases.

The cryoenzymology of several different beta-lactamases has been investigated. Particular attention has been paid to the experimental pitfalls of the technique. These include such factors as false bursts at the start of the reaction, instability of the enzymes during turnover, and Km values so high that little of the enzyme is present as a complex. Many of the difficulties in cryoenzymology stem from the use of organic cryosolvents. A novel "salt" cryosolvent has been tested: ammonium acetate solutions can be used down to about -60 degrees C. The enzymes examined are readily soluble, and stable, in this solvent. Nevertheless, out of 17 beta-lactamase beta-lactam systems, only 4 proved suitable for detailed investigation. In two of these, the hydrolysis of nitrocefin or 7-(thienyl-2-acetamido)-3-[[2-[[4- (dimethylamino)phenyl]azo]pyridinio]-methyl]cephem-4-carboxylic acid (PADAC), by beta-lactamase I from Bacillus cereus, substrate was converted into product at a slow enough rate (at -60 or -55 degrees C, respectively) for it to be possible to do successive scans during the course of the reaction. The spectra were those of substrate and product, and no intermediate was detected. The results argue against the accumulation of intermediate acyl-enzyme. The hydrolysis of PADAC by the P99 beta-lactamase from Enterobacter cloacae again showed spectra characteristic of substrate and product, and there was, moreover, a break in the Arrhenius plot; it is possible that a conformational change is (at least partially) rate-determining. The hydrolysis of dinitrophenylpenicillin by the P99 beta-lactamase did show features suggesting the accumulation of acyl-enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus cereus↗

PubMed1987

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.

Anti-Bacterial Agents↗

PubMed1987

An explicit model for bacterial resistance: application to beta-lactam antibiotics.

The effectiveness of beta-lactam antibiotics against Gram-negative bacteria that contain beta-lactamases is considered in terms of three factors. These are a rate constant for reaction of the beta-lactam antibiotic with a transpeptidase, the kinetic parameters for hydrolysis of the antibiotic by the beta-lactamase, and the permeability of the outer membrane. The concept of a dimensionless permeability number (Pn) is developed. When the permeability number is much less than one then permeability is important, and the MIC may be raised by a factor of up to 1/Pn. When the antibiotic is sufficiently reactive its effectiveness is given by the 'reactivity-permeability' product.

Anti-Bacterial Agents↗

PubMed1986

Carboxy groups as essential residues in beta-lactamases.

Beta-lactamases are divided into classes A, B and C on the basis of their amino acid sequences. Beta-Lactamases were incubated at pH 4.0 with the carboxy-group reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide plus a coloured nucleophile and the extents of inactivation and nucleophile incorporation were monitored. Two class A enzymes (from Bacillus cereus and Bacillus licheniformis) and two class C enzymes (from Enterobacter cloacae P99 and Pseudomonas aeruginosa) were examined. All four enzymes were inactivated, with total inactivation corresponding to the incorporation of approx. 2-3 mol of nucleophile/mol of enzyme. In the case of beta-lactamase I from Bacillus cereus, some 53% of the incorporated nucleophile was located on glutamic acid-168 in the amino acid sequence.

Amino Acid Sequence↗

PubMed1986

Changes in the coordination geometry of the active-site metal during catalysis of benzylpenicillin hydrolysis by Bacillus cereus beta-lactamase II.

Rapid-scanning stopped-flow spectroscopy (425-700 nm) has been used to study spectral changes in cobalt(II)-substituted Bacillus cereus beta-lactamase II during the binding and hydrolysis of benzylpenicillin. The experiments were carried out in aqueous solution over a temperature range of 3-20 degrees C. Three metallointermediates have been characterized by their visible absorption spectra. Two of them have visible absorption spectra identical with the intermediates ES1 and ES2 previously observed at subzero temperatures in a mixed aqueous/organic solvent [Bicknell, R., & Waley, S.G. (1985) Biochemistry 24, 6876-6887]. In addition, the branched kinetic pathway observed with the zinc(II) and cobalt(II) beta-lactamase II at subzero temperatures has been shown to occur with the cobalt(II)-substituted enzyme in aqueous solution at above-zero temperatures; thus, at pH 6.0 and 3 degrees C, the rate and equilibrium constants are readily determined for the reaction scheme: (Formula: see text). A third transient intermediate (called ES*) was found to precede ES1 in the pre-steady-state time period. The identity of the intermediates formed in aqueous solution with those previously observed in the cryostudy confirms that the mechanism is not changed either by the presence of an organic cosolvent or by subzero temperatures. Further characterization of ES1 and the steady-state intermediate ES2 at subzero temperatures, where their lifetime may be extended for up to several hours, has involved circular and magnetic circular dichroic studies. The magnetic circular dichroic spectra identify changes in the coordination sphere of the active-site metal during catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus cereus↗

PubMed1986

6-beta-Iodopenicillanate as a probe for the classification of beta-lactamases.

An inactivator of serine beta-lactamases, 6 beta-iodopenicillanate, can be utilized as a probe in the classification of beta-lactamases. It is a substrate for class-B Zn2+-containing beta-lactamase II. Although it inactivates enzymes from both classes A and C, it is much more efficient for the former group, with which it sometimes interacts following a branched pathway. On the basis of these observations, predictions are made concerning the class to which several enzymes belong.

Hydrogen-Ion Concentration↗

PubMed1986

The determination of specificity constants in enzyme-catalysed reactions.

A convenient and accurate procedure for determining the kinetic parameter Vmax./Km is described. This avoids the error in the usual method of taking the observed first-order rate constant of an enzymic reaction at low substrate concentration as Vmax./Km. A series of reactions is used in which the initial concentration of substrate is below Km (e.g. from 5% to 50% of Km). Measurements are taken over the same extent of reaction (e.g. 70%) for each member of the series, and treated as if the kinetics were truly first-order. The reciprocal of the observed first-order rate constant is then plotted against the initial concentration of substrate: the reciprocal of the ordinate intercept is Vmax./Km. The procedure, as well as being applicable to simple reactions, is shown to be valid when there is competitive inhibition by the product, or when the reaction is reversible, or when there is competitive or mixed inhibition. The hydrolysis of cephalosporin C by a beta-lactamase from Pseudomonas aeruginosa is used to illustrate the method.

Enzyme Inhibitors↗

PubMed1986

Structural and kinetic studies on beta-lactamase K1 from Klebsiella aerogenes.

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.

Amino Acid Sequence↗

PubMed1986

Identification of an essential glutamic acid residue in beta-lactamase II from Bacillus cereus.

Beta-Lactamase II from Bacillus cereus was readily inactivated by incubation at pH 4.75 with a water-soluble carbodiimide plus a suitable nucleophile. In the early stages of the reaction, 1 equivalent of nucleophile was incorporated/equivalent of enzyme, whereas during the later stages a second equivalent of nucleophile was also incorporated. This latter process correlated with the blocking of the enzyme's single thiol group. Enzyme inactivated in the presence of the coloured nucleophile N-(2,4-dinitrophenyl)ethylenediamine was fragmented by pepsin digestion, and coloured peptides were isolated by gel filtration and h.p.l.c. Two major peptides, representing 52% of the incorporated label, were isolated and sequenced. Both peptides contained the incorporated label on glutamic acid-37, and it is concluded that this latter residue represents a catalytically essential carboxylic residue in beta-lactamase II.

Bacillus cereus↗

PubMed1985

Cryoenzymology of Bacillus cereus beta-lactamase II.

The effects of cryosolvents and subzero temperatures on the metalloenzyme beta-lactamase II from Bacillus cereus have been investigated. Preliminary experiments led to the selection of suitable systems for the study of beta-lactamase II catalysis at low temperatures, namely, cobalt(II) beta-lactamase II hydrolysis of benzylpenicillin in 60% (v/v) ethylene glycol and zinc beta-lactamase II hydrolysis of the chromophoric cephalosporin nitrocefin in 60% (v/v) methanol. Progress curves for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II in 60% (v/v) ethylene glycol at temperatures below -30 degrees C consisted of a transient followed by a steady-state phase. The amplitude of the transient implied a burst whose magnitude was greater than the concentration of enzyme, and the proposed mechanism comprises a branched pathway. The kinetics for the simplest variants of such pathways have been worked out, and the rate constants (and activation parameters) for the individual steps have been determined. The spectrum of the enzyme changed during turnover: when benzylpenicillin was added to cobalt beta-lactamase II, there was a large increase in the cysteine-cobalt(II) charge-transfer absorbance at 333 nm. This increase occurred within the time of mixing, even at -50 degrees C. The subsequent decrease in A333 was characterized by a rate constant that had the same value as the "branching" rate constant of the branched-pathway mechanism. This step is believed to be a change in conformation of the enzyme-substrate complex. Single-turnover experiments utilized the change in A333, and the results were consistent with pre-steady-state and steady-state experiments. When a single-turnover experiment at -48 degrees C was quenched with acid, the low molecular weight component of the intermediate was shown to be substrate. The mechanism advanced for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II involves two noncovalent enzyme-substrate complexes that have been characterized by their electronic absorption spectra. When manganese beta-lactamase II was used, the same features (implying a branched pathway) were evident; these experiments were carried out at ordinary temperatures and did not utilize a cryosolvent. The hydrolysis of nitrocefin by zinc beta-lactamase II has been studied concurrently in 60% (v/v) methanol. Progress curves were triphasic. There were two transients preceding the linear steady-state phase. The stoichiometry of the burst again implied a branched pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacillus cereus↗

PubMed1985

Single-turnover and steady-state kinetics of hydrolysis of cephalosporins by beta-lactamase I from Bacillus cereus.

The kinetics of the hydrolysis of two cephalosporins by beta-lactamase I from Bacillus cereus 569/H/9 has been studied by single-turnover and steady-state methods. Single-turnover kinetics could be measured over the time scale of minutes when cephalosporin C was the substrate. The other substrate, 7-(2',4'-dinitrophenylamino)deacetoxycephalosporanic acid, was hydrolysed even more slowly, and has potential for use in crystallographic studies of beta-lactamases. Comparison of single-turnover and steady-state kinetics showed that, for both substrates, opening the beta-lactam ring (i.e. acylation of the enzyme) was the rate-determining step. Thus the non-covalent enzyme-substrate complex is expected to be the intermediate observed crystallographically.

Ammonium Sulfate↗

PubMed1985

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

The amino acid sequence of the zinc-requiring beta-lactamase 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 beta-lactamase, and is structurally and mechanistically distinct from both the widely distributed class A beta-lactamases (such as the Escherichia coli RTEM enzyme) and from the chromosomally encoded class C enzymes from Gram-negative bacteria.

Amino Acid Sequence↗

PubMed1985

The production and molecular properties of the zinc beta-lactamase of Pseudomonas maltophilia IID 1275.

The production and purification of a tetrameric zinc beta-lactamase from Pseudomonas maltophilia IID 1275 were greatly improved. Three charge variants were isolated by chromatofocusing. The subunits each contain two atomic proportions of zinc and (in two of the variants) one residue of cysteine. The thiol group is not required for activity, nor does it appear to bind to the metal. Replacement of zinc by cobalt, cadmium or nickel takes place at a measurable rate, and gives enzymes that are less active than the zinc enzyme. The properties of this enzyme differ from those of the other known zinc beta-lactamase, beta-lactamase II from Bacillus cereus. The amino acid sequence of the N-terminal 32 residues was determined; there is no similarity to the N-terminal sequences of other beta-lactamases.

Amino Acids↗

PubMed1985

Kinetics of suicide substrates. Practical procedures for determining parameters.

Many clinically important or mechanistically interesting inhibitors react with enzymes by a branched pathway in which inactivation of the enzyme and formation of product are competing reactions. The steady-state kinetics for this pathway [Waley (1980) Biochem. J. 185, 771-773] gave equations for progress curves that were cumbersome. A convenient linear plot is now described. The time (t1/2) for 50% inactivation of the enzyme (this is also the time for 50% formation of product), or for 50% loss of substrate, is measured in a series of experiments in which the concentration of inhibitor, [I]0, is varied; in these experiments the ratio of the concentration of enzyme to the concentration of inhibitor is kept fixed. Then a plot of [I]0 X t1/2 against [I]0 is linear, and the kinetic parameters can be found from the slope and intercept. Furthermore, simplifications of the equations for progress curves are described that are valid when the concentration of inhibitors is high, or is low, or when the extent of reaction is low. The use of simulated data has shown that the recommended methods are not unduly sensitive to experimental error.

Enzyme Inhibitors↗

PubMed1985

Inhibition of class C beta-lactamases by (1'R,6R)-6-(1'-hydroxy)benzylpenicillanic acid SS-dioxide.

beta-Lactamases, enzymes that catalyse the hydrolysis of the beta-lactam ring in beta-lactam antibiotics, are divided into three classes, A, B and C, on the basis of the structures so far determined. There are relatively few effective inhibitors of class C beta-lactamases. A beta-lactam sulphone with a hydroxybenzyl side chain, namely (1'R,6R)-6-(1'-hydroxy)benzylpenicillanic acid SS-dioxide (I), has now been studied. The sulphone is a good mechanism-based inhibitor of class C beta-lactamases. At pH8, the inhibition of a Pseudomonas beta-lactamase is irreversible, and proceeds at a rate that is about one-tenth the rate of concurrent hydrolysis. The labelled enzyme has enhanced u.v. absorption and is probably an enamine. At a lower pH, however, inhibition is transitory.

Cephalosporins↗

PubMed1984

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.

Amino Acids↗