Inhibition of Class D β-Lactamases by Diaroyl Phosphates

• Published in: Biochemistry (Easton) 2005-12, Vol.44 (49), p.16121-16129
• Main Authors: Majumdar, Sudipta, Adediran, S. A, Nukaga, Michiyoshi, Pratt, R. F
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - metabolism; beta-Lactamase Inhibitors; beta-Lactamases - metabolism; Binding Sites; Isoenzymes - antagonists & inhibitors; Isoenzymes - metabolism; Molecular Structure; Phosphates - chemistry; Phosphates - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi051719s

The production of β-lactamases is an important component of bacterial resistance to β-lactam antibiotics. These enzymes catalyze the hydrolytic destruction of β-lactams. The class D serine β-lactamases have, in recent years, been expanding in sequence space and substrate spectrum under the challenge of currently dispensed β-lactams. Further, the β-lactamase inhibitors now employed in medicine are not generally effective against class D enzymes. In this paper, we show that diaroyl phosphates are very effective inhibitory substrates of these enzymes. Reaction of the OXA-1 β-lactamase, a typical class D enzyme, with diaroyl phosphates involves acylation of the active site with departure of an aroyl phosphate leaving group. The interaction of the latter with polar active-site residues is most likely responsible for the general reactivity of these molecules with the enzyme. The rate of acylation of the OXA-1 β-lactamase by diaroyl phosphates is not greatly affected by the electronic effects of substituents, probably because of compensation phenomena, but is greatly enhanced by hydrophobic substituents; the second-order rate constant for acylation of the OXA-1 β-lactamase by bis(4-phenylbenzoyl) phosphate, for example, is 1.1 × 107 s-1 M-1. This acylation reactivity correlates with the hydrophobic nature of the β-lactam side-chain binding site of class D β-lactamases. Deacylation of the enzyme is slow, e.g., 1.24 × 10-3 s-1 for the above-mentioned phosphate and directly influenced by the electronic effects of substituents. The effective steady-state inhibition constants, K i, are nanomolar, e.g., 0.11 nM for the above-mentioned phosphate. The diaroyl phosphates, which have now been shown to be inhibitory substrates of all serine β-lactamases, represent an intriguing new platform for the design of β-lactamase inhibitors.