Investigation of the covalent modification of the catalytic triad of human cytomegalovirus protease by pseudo-reversible β-lactam inhibitors and a peptide chloromethylketone

Autor:	Christina Bessant, Cathy A. van Wely, Douglas S. Montgomery, Alan D. Borthwick, Graham J. Hart, Ian J. Purvis, Terry M. Haley, S. Jane Angier, Devi H. Smart
Rok vydání:	1998
Předmět:	chemistry.chemical_classification Protease biology Chemistry Stereochemistry Electrospray ionization medicine.medical_treatment Active site Peptide Mass spectrometry Tandem mass spectrometry Trypsin Biochemistry Catalytic triad biology.protein medicine Spectroscopy medicine.drug
Zdroj:	Journal of Mass Spectrometry. 33:1246-1255
ISSN:	1096-9888 1076-5174
Popis:	An investigation into the interaction between human cytomegalovirus (HCMV) protease and several β-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 β-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 identification of the site of covalent modification and confirmed 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 confirmed His 63 as the active site imidazole nucleophile. © 1998 John Wiley & Sons, Ltd.
Databáze:	OpenAIRE
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