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Investigation of the covalent modification of the catalytic triad of human cytomegalovirus protease by pseudo‐reversible β‐lactam inhibitors and a peptide chloromethylketone
Author(s) -
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.
Publication year - 1998
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/(sici)1096-9888(199812)33:12<1246::aid-jms743>3.0.co;2-e
Subject(s) - chemistry , protease , electrospray ionization , peptide , active site , trypsin , mass spectrometry , chromatography , tandem mass spectrometry , serine protease , enzyme , biochemistry , combinatorial chemistry , stereochemistry
An investigation into the interaction between human cytomegalovirus(HCMV) protease and several β‐lactams, withcharacterization of the resulting acylenzymes using mass spectrometry,is reported. The time dependence of the inhibitors is highlighted bymaking comparisons of values obtained for inhibition and acylation.Analysis of inactivated HCMV protease revealed aβ‐lactam:protease stoichiometry of 1. Subsequent enzymaticdigestion with trypsin, peptide mapping using liquid chromatographycoupled with electrospray ionization mass spectrometry and sequencingby nanoelectrospray tandem mass spectrometry(NanoES‐MS/MS) allowed the identification of thesite of covalent modification and confirmed Ser 132 as the active sitehydroxyl nucleophile. Further, treatment of the protease with apeptide chloromethylketone and sequence analysis usingNanoES‐MS/MS of the alkylated enzyme confirmed His 63 asthe active site imidazole nucleophile. © 1998 John Wiley &Sons, Ltd.