Identification of Active-Site Residues of the Adenovirus Endopeptidase

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 91; no. 3; pp. 844 - 847
• Main Authors: Rancourt, Claudine, Tihanyi, Karoly, Bourbonniere, Martin, Weber, Joseph M.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.02.1994; National Acad Sciences; National Academy of Sciences
• Subjects: Active sites; Adenoviridae - enzymology; Adenoviridae - genetics; Adenoviruses; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Binding Sites - genetics; Biological and medical sciences; Biology; Cattle; Cloning, Molecular; Cysteine - genetics; Cysteine - metabolism; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Dogs; Enzyme activity; Enzymes; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genetic mutation; Humans; Hydrolases; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Phosphates; Protein precursors; Proteins; Sequence Homology, Amino Acid; Species Specificity; Virions; Viruses; Virus; Conserved sequence; Adenoviridae; Enzyme; Interspecific comparison; Endopeptidase; Active site; Sequence alignment; Aminoacid sequence; Structural analysis
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.3.844

Multiple sequence alignment of the 12 adenovirus endopeptidases known to date identified a number of conserved residues which might be important for enzyme activity. Eleven mutants were created in the cloned gene by site-directed mutagenesis to identify the active site of this thiol endopeptidase. Analysis of the proteolytic activity in a crude system using viral precursor proteins, as well as in a purified system with activated proteinases using a new chromophoric octapeptide substrate, yielded results consistent with Cys-104 and His-54 being two members of the active site. This result was confirmed by the carboxymethylation of the reactive Cys-104 and its prevention by the active-thiol-specific agent E64. Although Cys-122 and Cys-126 were also reactive cysteines, mutation of these residues did not affect enzyme activity. Replacement of the active-site Cys-104 by serine converted the enzyme into a serine-like proteinase, sensitive to serine proteinase inhibitors. The absence of homology to other proteinases, particularly at the active-site cysteine, coupled with the requirement for activation by a substrate cleavage fragment, indicates that the adenovirus endoproteinase may represent a new subclass of cysteine proteinases.