Infrared Spectroscopy of Interactions Between Antibiotics and β-Lactamases/Transpeptidases

Abstract

The serine proteinase enzymes form a disparate family that encompasses the digestive enzymes chymotrypsin and trypsin, thrombin, which is involved in blood clotting, as well as bacterial enzymes such as subtilisin. The structures of the mammalian enzymes fall into one class and are composed predominantly of β-sheet, while the bacterial enzymes are predominantly α-helical. Despite this difference in structure all serine proteinases have an essentially identical catalytic apparatus. This is comprised of the relay system [Asp-His-Ser], the oxyanion hole and a specificity conferring sidechain binding pocket. The serine of the relay system acts as a nucleophile which attacks the substrate at the susceptible carbonyl group, where the peptide chain is cleaved. The His residue, strongly hydrogen bonded to the Ser acts as a general base to remove a proton from the Ser in both steps of the reaction (see below) and to provide a proton, while acting as an acid, for the leaving nitrogen atom in acylation or the serine in deacylation. The Asp residue serves to stabilise the conformation of the His residue as required for its action as a general base. The oxyanion hole is comprised of two backbone amide nitrogens, whose hydrogens are so placed to hydrogen bond to the susceptible carbonyl that is to be attacked by the serine residue. The predefined spatial organisation of these amide hydrogens ensures that there is no entropy requirement upon substrate or acyl group interaction. The oxyanion hole has been supposed to stabilise the negative charge which develops in the transition states for each of the two steps of the enzyme-catalysed reaction:where EA is the acylenzyme. The C-terminal part of the substrate is lost in the acylation step, characterised by k2, while the N-terminal part is lost as a consequence of deacylation, characterised by k3. The acylation step generates a unique ester bond in the structure of the covalently bonded enzyme substrate complex. The deacylation step represents a hydrolysis of the ester intermediate. The acylation and deacylation steps are almost mirror images of one another. The amide bonds of proteins or peptides are hydrolytically resistant and a complex mechanism has evolved to cope with this. The unique ester group of the acylenzyme is amenable to study using IR spectroscopy since it absorbs at a frequency that is higher than the peptide amide groups, although 13C=Oisotope labelling is required to ensure band assignment, since perturbation of the protein spectrum by formation of the acylenzyme can, at low pH, interfere with the ester band(s) [1–5].