Characterization of three components of human adenovirus proteinase activity in vitro

Human adenovirus contains a virion-associated proteinase activity essential for the development of infectious virus. Maximal proteinase activity in vitro had been shown to require three viral components: the L3 23-kDa protein, an 11-amino acid cofactor (pVIc), and the viral DNA. Here, we present a quantitative purification procedure for a recombinant L3 23-kDa protein (recombinant endoproteinase (rEP)) expressed in Escherichia coli and the procedure that led to the purification and identification of pVIc as a cofactor, The cofactors stimulate proteinase activity not by decreasing K-m, which changes by no more than 2-fold, but by increasing k(cat). rEP alone had a small amount of activity, the k(cat) of which increased 355-fold with pVIc and 6072-fold with adenovirus serotype 2 (Ad2) DNA as well, Curves of V-max of rEP . pVIc complexes with the substrate (Leu-Arg-Gly-Gly-NH)(2)-rhodamine as a function of pH in the absence and presence of Ad2 DNA indicate that the pK(alpha) values of amino acids that affect catalysis are quite different from those that affect catalysis by the cysteine proteinase papain, The pK(alpha), values in the absence of Ad2 DNA are 5.2, 6.4, 6.9, 7.5, and 9.4, and those in its presence are 5.2, 6.5, 7.4, and 8.8.