Investigation of the covalent modification of the catalytic triad of human cytomegalovirus protease by pseudo-reversible β-lactam inhibitors and a peptide chloromethylketone
✍ Scribed by Haley, Terry M.; Angier, S. Jane; Borthwick, Alan D.; Montgomery, Douglas S.; Purvis, Ian J.; Smart, Devi H.; Bessant, Christina; Van Wely, Cathy; Hart, Graham J.
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 162 KB
- Volume
- 33
- Category
- Article
- ISSN
- 1076-5174
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✦ Synopsis
An investigation into the interaction between human cytomegalovirus (HCMV) protease and several b-lactams, with characterization of the resulting acylenzymes using mass spectrometry, is reported. The time dependence of the inhibitors is highlighted by making comparisons of values obtained for inhibition and acylation. Analysis of inactivated HCMV protease revealed a b-lactam : protease stoichiometry of 1. Subsequent enzymatic digestion with trypsin, peptide mapping using liquid chromatography coupled with electrospray ionization mass spectrometry and sequencing by nanoelectrospray tandem mass spectrometry (NanoES-MS/MS) allowed the identiÐcation of the site of covalent modiÐcation and conÐrmed Ser 132 as the active site hydroxyl nucleophile. Further, treatment of the protease with a peptide chloromethylketone and sequence analysis using NanoES-MS/MS of the alkylated enzyme conÐrmed His 63 as the active site imidazole nucleophile.