A POINT MUTATION LEADS TO ALTERED PRODUCT SPECIFICITY IN BETA-LACTAMASE CATALYSIS

beta-Lactamases are the primary cause of beta-lactam antibiotic resist ance in many pathogenic organisms. The beta-lactamase catalytic mechan ism has been shown to involve a covalent acyl-enzyme. Examination of t he structure of the class A beta-lactamase from Bacillus licheniformis suggested that replacement of Asn-170 by leucine would disrupt the de acylation reaction by displacing the hydrolytic water molecule. When N 170L beta-lactamase was reacted with penicillins, a novel product was formed. We postulate that with leucine at position 170 the acyl-enzyme undergoes deacylation by an intramolecular rearrangement (rather than hydrolysis) to form a thiazolidine-oxazolinone as the initial product . The oxazolinone subsequently undergoes rapid breakdown leading to th e formation of N-phenylacetylglycine and N-formylpenicillamine. This a ppears to be the first reported case where a point mutation leads to a change in enzyme mechanism resulting in a substantially altered produ ct, effectively changing the product specificity of beta-lactamase int o that of D-Ala-D-Ala-carboxypeptidase interacting with benzylpenicill in.