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An investigation into the mechanisms of action of #beta#-lactamase enzymes 1 and 2 from B. cereus 569/H

By David G. Proctor and ICI pharmaceuticals


The pH dependence of the B-lactamase enzymes I and 2 catalysed hydrolysis of\ud penicillin and cephalosporin derivatives has been investigated. It was shown that the Blactamase\ud I catalysed hydrolysis of n-alkyl penicillins of increasing chain length produced\ud an increase inkcaVKm up to the octyl derivative. Despite this it is believed that the activesite\ud of the B-lactamase I enzyme does not display a strong recognition for B-lactams\ud containing a hydrophobic C6 side-chain.\ud \ud \ud It was found that the pH dependence for the enzyme catalysed hydrolysis of\ud benzyl penicillin and benzyl cephalosporin did not decline as expected at low pH, but\ud instead the rate levels off.\ud \ud \ud The incorporation of a negatively charged group into the phenyl C7 side chain of\ud cephalosporins resulted in lower activity at pH7 compared to that for benzyl cephalosporin,\ud but at low pH much higher activity is seen: 2-carboxyphenyl cephalosporin was 10 fold more\ud reactive at pH3 than at pH7. The B-lactamase I catalysed hydrolysis of phenyl substituted\ud penicillins containing a negatively charged functional group in the phenyl side chain was\ud more complex. 2-Carboxyphenyl penicillin shows higher activity at low pH, while the 3,4-\ud carboxyphenyl penicillin derivatives show a typical 'bell-shaped' profile, but with the pKa I\ud value shifted up-field. This was further investigated by preparing the cis/trans isomers of 2-\ud carboxycyclohexyl penicillin and cephalosporin. \ud \ud \ud It was found that the cis 2-\ud carboxycyclohexyl penicillin compound displayed high activity at low pH, while the trans\ud compound did not. Neither of the corresponding cepbalosporin derivatives showed evidence\ud of higher activity at low pH.\ud \ud \ud The C3 ester and C3 alcohol of penicillins and the C4 cephalosporin lactone were\ud synthesised. The penicillin alcohol and the cephalosporin lactone were found to be 'good'\ud substrates for the B-lactamase I enzyme and produced 'normal' pH-rate profiles.\ud \ud \ud It was found that with the B-lactamase 2 enzyme hydrolysis of n-alkyl penicillins\ud proceeded at comparable rates irrespective of the length of the C6 side chain alkyl group,\ud and that the enzyme hydrolysis rate at pH 7 was minimally affected by substituent changes\ud in the C6/C7 side chains of penicillin and cephalosporin but the behaviour was more\ud complex when the negatively charged carboxyphenyl B-lactams were investigated. The\ud enzyme catalysed hydrolysis was greatly reduced when the C3/4 carboxylate group of the\ud substrate was converted into an alcohol, ester or lactone, suggesting the importance of this\ud group in the 13-lactamase2 hydrolysis mechanism.\ud \ud \ud These results raise important questions about the the existing hypotheses which\ud attempt to explain the mechanism of action of the B-lactamase I and 2 enzymes

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