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.