title: The protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis.
abstract: Sequence analysis and site-directed mutagenesis were used to study the mechanisms of activation and catalysis of the adenovirus type 2 (Ad2) protease. Primary structure alignments of proteases from 12 serotypes and previously elucidated inhibition profiles were used to target residues for mutagenesis. All conserved serine and cysteine residues were mutated separately and following expression in Escherichia coli their activity in a synthetic peptide assay was compared to that of wild-type recombinant protease. Mutants containing altered serine residues were active while mutations to cysteine-104 and cysteine-122 reduced activity by more than 95%. These results taken together with the known inhibition profile of the adenovirus protease confirm that it is a cysteine protease and suggest that one of these residues provides the active site nucleophile while the other is a part of the thiol-disulphide interchange mechanism previously reported to be involved in its activation.
authors.affiliation: Division of Cell and Molecular Biology, School of Biological and Medical Sciences, University of St Andrews, U.K.
authors.forename: A W
authors.initials: AW
authors.lastname: Grierson
authors.forename: R
authors.initials: R
authors.lastname: Nicholson
authors.forename: P
authors.initials: P
authors.lastname: Talbot
authors.forename: A
authors.initials: A
authors.lastname: Webster
authors.forename: G
authors.initials: G
authors.lastname: Kemp
mesh_terms: D000260:Adenoviruses, Human / Q000145:classification / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine; D010450:Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D017921:Mastadenovirus / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012703:Serotyping; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D010450:Endopeptidases; D003545:Cysteine