Proctor, David G. (1991) An investigation into the mechanisms of action of #beta#-lactamase enzymes 1 and 2 from B. cereus 569/H. Doctoral thesis, Huddersfield Polytechnic.

The pH dependence of the B-lactamase enzymes I and 2 catalysed hydrolysis of
penicillin and cephalosporin derivatives has been investigated. It was shown that the Blactamase
I catalysed hydrolysis of n-alkyl penicillins of increasing chain length produced
an increase inkcaVKm up to the octyl derivative. Despite this it is believed that the activesite
of the B-lactamase I enzyme does not display a strong recognition for B-lactams
containing a hydrophobic C6 side-chain.

It was found that the pH dependence for the enzyme catalysed hydrolysis of
benzyl penicillin and benzyl cephalosporin did not decline as expected at low pH, but
instead the rate levels off.

The incorporation of a negatively charged group into the phenyl C7 side chain of
cephalosporins resulted in lower activity at pH7 compared to that for benzyl cephalosporin,
but at low pH much higher activity is seen: 2-carboxyphenyl cephalosporin was 10 fold more
reactive at pH3 than at pH7. The B-lactamase I catalysed hydrolysis of phenyl substituted
penicillins containing a negatively charged functional group in the phenyl side chain was
more complex. 2-Carboxyphenyl penicillin shows higher activity at low pH, while the 3,4-
carboxyphenyl penicillin derivatives show a typical 'bell-shaped' profile, but with the pKa I
value shifted up-field. This was further investigated by preparing the cis/trans isomers of 2-
carboxycyclohexyl penicillin and cephalosporin.

It was found that the cis 2-
carboxycyclohexyl penicillin compound displayed high activity at low pH, while the trans
compound did not. Neither of the corresponding cepbalosporin derivatives showed evidence
of higher activity at low pH.

The C3 ester and C3 alcohol of penicillins and the C4 cephalosporin lactone were
synthesised. The penicillin alcohol and the cephalosporin lactone were found to be 'good'
substrates for the B-lactamase I enzyme and produced 'normal' pH-rate profiles.

It was found that with the B-lactamase 2 enzyme hydrolysis of n-alkyl penicillins
proceeded at comparable rates irrespective of the length of the C6 side chain alkyl group,
and that the enzyme hydrolysis rate at pH 7 was minimally affected by substituent changes
in the C6/C7 side chains of penicillin and cephalosporin but the behaviour was more
complex when the negatively charged carboxyphenyl B-lactams were investigated. The
enzyme catalysed hydrolysis was greatly reduced when the C3/4 carboxylate group of the
substrate was converted into an alcohol, ester or lactone, suggesting the importance of this
group in the 13-lactamase2 hydrolysis mechanism.

These results raise important questions about the the existing hypotheses which
attempt to explain the mechanism of action of the B-lactamase I and 2 enzymes.

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