Investigation of the covalent modification of the catalytic triad of human cytomegalovirus protease by pseudo-reversible beta-lactam inhibitors and a peptide chloromethylketone.
An investigation into the interaction between human cytomegalovirus (HCMV) protease and several beta-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 beta-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.
Haley
T M
TM
Glaxo Wellcome Research & Development, Medicines Research Centre, Stevenage, Hertfordshire, UK. tmh0149@ggr.co.uk
Angier
S J
SJ
Borthwick
A D
AD
Montgomery
D S
DS
Purvis
I J
IJ
Smart
D H
DH
Bessant
C
C
Van Wely
C
C
Hart
G J
GJ
D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000201:enzymology* / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D014357:Trypsin; D047090:beta-Lactams / Q000737:chemistry / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D004279:DNA, Viral; D011480:Protease Inhibitors; D047090:beta-Lactams; D012697:Serine Endopeptidases; C071910:assemblin; D014357:Trypsin