Affiliation:
1. School of Biochemistry, University of Birmingham, Birmingham B15 2TT, United Kingdom
2. Preclinical Science, Pharma Division, F. Hoffmann-La Roche Ltd, Basel CH-4070, Switzerland
Abstract
The serine proteinase mechanism has been studied using a wide range of techniques over
many years and is now well understood in terms of the formal chemical changes that
occur on the reaction pathway. At the atomic level our understanding is less secure in
that available techniques are unable to define interactions such as hydrogen bonding
with sufficient accuracy. Atomic interaction is strongly dependent upon separation
distances and these need to be measured either directly or indirectly in the dynamic
reacting system. Infrared spectroscopy has been applied to the study of chymotrypsin
acylenzyme reaction intermediates with the aim of measuring, albeit indirectly, the
strength of hydrogen bonding in the oxyanion hole catalytic device. These measurements
have been successful with moderately specific substrates but there is a long way to go in
terms of improved time-resolution. It is tentatively proposed that tetrahedral
intermediates accumulate at high pH. This is, we believe, the first report of the relatively
direct observation of this phenomenon in any reacting ester system, chemical or enzymic.
The approach used with the serine proteinases has been applied to studies of the
transpeptidase of Streptococcus pneumoniae
PBP2x. We have shown that the acylenzyme
formed from benzylpenicillin hydrogen bonds most strongly in the oxyanion hole. This
bonding, in contrast to serine proteinases and β-lactamases where the interaction
facilitates catalysis, serves to stabilise the ester intermediate as required for effective
antibiotic action. We see this as a good example of ‘Nature knows Best’ since semisynthetic
antibiotics, designed to be resistant to hydrolysis by β-lactamases, hydrogen
bond more weakly and the acylenzymes hydrolyse more rapidly.
Subject
Spectroscopy,Biochemistry,Atomic and Molecular Physics, and Optics
Cited by
5 articles.
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