IDENTIFICATION OF ACTIVE-SITE RESIDUES OF THE ADENOVIRUS ENDOPEPTIDASE

Multiple sequence alignment of the 12 adenovirus endopeptidases known to date identified a number of conserved residues which might be impor tant for enzyme activity. Eleven mutants were created in the cloned ge ne by site-directed mutagenesis to identify the active site of this th iol endopeptidase. Analysis of the proteolytic activity in a crude sys tem using viral precursor proteins, as well as in a purified system wi th activated proteinases using a new chromophoric octapeptide substrat e, yielded results consistent with Cys-104 and His-54 being two member s of the active site. This result was confirmed by the carboxymethylat ion of the reactive Cys-104 and its prevention by the active-thiol-spe cific agent E64. Although Cys-122 and Cys-126 were also reactive cyste ines, mutation of these residues did not affect enzyme activity. Repla cement of the active-site Cys-104 by serine converted the enzyme into a serine-like proteinase, sensitive to serine proteinase inhibitors. T he absence of homology to other proteinases, particularly at the activ e-site cysteine, coupled with the requirement for activation by a subs trate cleavage fragment, indicates that the adenovirus endoproteinase may represent a new subclass of cysteine proteinases.