A soluble form of Streptococcus pneumoniae PBP2x, a molecular target of penicillin and cephalosporin antibiotics, has been expressed and purified. IR difference spectra of PBP2x acylated with benzylpenicillin, cloxacillin, cephalothin and ceftriaxone have been measured. The difference spectra show two main features. The ester carbonyl vibration of the acyl-enzyme is ascribed to a small band between 1710 and 1720 cm-1, whereas a much larger band at approx. 1640 cm-1 is ascribed to a perturbation in the structure of the enzyme, which occurs on acylation. The protein perturbation has been interpreted as occurring in beta-sheet. The acyl-enzyme formed with benzylpenicillin shows the lowest ester carbonyl vibration frequency, which is interpreted to mean that the carbonyl oxygen is the most strongly hydrogen-bonded in the oxyanion hole of the antibiotics studied. The semi-synthetic penicillin cloxacillin is apparently less well organized in the active site and shows two partially overlapping ester carbonyl bands. The penicillin acyl-enzyme has been shown to deacylate more slowly than that formed with cloxacillin. This demonstrates that the natural benzylpenicillin forms a more optimized and better-bonded acyl-enzyme and that this in turn leads to the stabilization of the acyl-enzyme required for effective action in the inhibition of PBP2x. The energetics of hydrogen bonding in the several acyl-enzymes is discussed and comparison is made with carbonyl absorption frequencies of model ethyl esters in a range of organic solvents. A comparison of hydrolytic deacylation with hydroxaminolysis for both chymotryspin and PBP2x leads to the conclusion that deacylation is uncatalysed.

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.

This paper describes a method by which the activity of an immobilized enzyme can be modulated electrochemically at an electrode. The particular example studied, involving the enzyme firefly luciferase being immobilized in a gelatin film of thickness <1 μm, provides a useful model system since changes in the catalytic activity can be measured instantaneously through the natural bioluminescent emission. Using this biointerfacial arrangement, we have been able to demonstrate the reversible switching off and on of the enzyme's activity. Through a series of mechanistic studies, we have been able to determine that the bioluminescence response is modulated (on long time scales) as a consequence of the electrochemical depletion of protons at the electrode interface resulting in a local increase in pH.

A soluble form of Streptococcus pneumoniae PBP2x, a molecular target of penicillin and cephalosporin antibiotics, has been expressed and purified. IR difference spectra of PBP2x acylated with benzylpenicillin, cloxacillin, cephalothin and ceftriaxone have been measured. The difference spectra show two main features. The ester carbonyl vibration of the acyl-enzyme is ascribed to a small band between 1710 and 1720 cm-1, whereas a much larger band at approx. 1640 cm-1 is ascribed to a perturbation in the structure of the enzyme, which occurs on acylation. The protein perturbation has been interpreted as occurring in beta-sheet. The acyl-enzyme formed with benzylpenicillin shows the lowest ester carbonyl vibration frequency, which is interpreted to mean that the carbonyl oxygen is the most strongly hydrogen-bonded in the oxyanion hole of the antibiotics studied. The semi-synthetic penicillin cloxacillin is apparently less well organized in the active site and shows two partially overlapping ester carbonyl bands. The penicillin acyl-enzyme has been shown to deacylate more slowly than that formed with cloxacillin. This demonstrates that the natural benzylpenicillin forms a more optimized and better-bonded acyl-enzyme and that this in turn leads to the stabilization of the acyl-enzyme required for effective action in the inhibition of PBP2x. The energetics of hydrogen bonding in the several acyl-enzymes is discussed and comparison is made with carbonyl absorption frequencies of model ethyl esters in a range of organic solvents. A comparison of hydrolytic deacylation with hydroxaminolysis for both chymotryspin and PBP2x leads to the conclusion that deacylation is uncatalysed.

Measurements are presented of the radiation inactivation of four enzymes exposed to a 6 MeV proton beam. It has long been thought that the measurement of the susceptibility of an enzyme to ionizing radiation can be used to determine its molecular mass. Results are frequently interpreted using the empirical analysis of Kempner and Macey (Biochim. Biophys. Acta 163, 188-203, 1963). We examine this analysis and discuss the validity and limitations of the assumptions on which it is based. Our results indicate that the specific biochemical properties of each enzyme make a significant contribution to its radiation sensitivity.