The mechanism of catalysis and the inhibition of β-lactamases

Abstract

Formation of a tetrahedral intermediate by nucleophilic attack on the β-lactam carbonyl carbon of penicillins generates a lone pair on the β-lactam nitrogen which is syn to the incoming nucleophile, in contrast to the normal anti arrangement found in peptides. Ring opening of the β-lactam requires protonation of the β-lactam nitrogen by a general acid catalyst. The general acid/base catalyst in β-lactamases is probably a glutamate and a tyrosine residue in class A and C enzymes, respectively. Phosphonamidates inactivate class C β-lactamases by phosphonylation of the active site serine, the rate of which is enhanced by a factor of at least 10⁶. The enzyme’s catalytic machinery used for hydrolysis is also used for phosphonylation. The rate enhancement may be greater than 10⁹ if the mechanism occurs by an inhibitor assisted reaction involving intramolecular general acid catalysis. Class B metallo-β-lactamases are inhibited by thiol derivatives with K_i as low as 10 µM. The mechanism of hydrolysis of the metallo-β-lactamase involves a dianionic tetrahedral intermediate stabilised by zinc(II).