The roles of the carboxy group in β-lactam antibiotics and lysine 234 in β-lactamase I

Abstract

The replacement of the C3 carboxylate in phenoxymethylpenicillin by a hydroxymethyl group and of the C4 carboxylate in cephalosporins by both a lactone and an aldehyde gives derivatives which are still good substrates for Bacillus cereus 569/H β-lactamase I. The enzyme rate-enhancement factors for the hydrolysis of the modified β-lactams vary from 10⁴ to 10⁶. All three modified substrates show bell-shaped (k_cat/K_m)–pH profiles indicative of two catalytically important ionising residues on the protein of pK_a, about 5 and 9. Although lysine 234 is a highly conserved residue in class A β-lactamases and has been traditionally thought to interact with the carboxylate of the β-lactam antibiotic, it is not responsible for the decrease in enzyme activity at high pH corresponding to the pK_aof about 9.