Identification of active-site residues of the adenovirus endopeptidase.

Abstract

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.