Interaction of the human adenovirus proteinase with its 11-amino acid cofactor pVIc

The interaction of the human adenovirus proteinase (AVP) and AVP-DNA comple xes with the 11-amino acid cofactor pVIc was characterized. The equilibrium dissociation constant for the binding of pVIc to AVP was 4.4 muM. The bind ing of AVP to 12-mer single-stranded DNA decreased the K-d for the binding of pVIc to AVP to 0.09 muM. The pVIc-AVP complex hydrolyzed the substrate w ith a Michaelis constant (K-m) of 3.7 muM and a catalytic rate constant (k( cat)) of 1.1 s(-1). In the presence of DNA, the K-m increased less than 2-f old, and the k(cat) increased 3-fold. Alanine-scanning mutagenesis was perf ormed to determine the contribution of individual pVIc side chains in the b inding and stimulation of AVP. Two amino acid residues, Gly1' and Phe11', w ere the major determinants in the binding of pVIc to AVP, while Val2' and P he11' were the major determinants in stimulating enzyme activity. Binding o f AVP to DNA greatly suppressed the effects of the alanine substitutions on the binding of mutant pVIcs to AVP. Binding of either or both of the cofac tors, pVIc, or the viral DNA, to AVP did not dramatically alter its seconda ry structure as determined by vacuum ultraviolet circular dichroism. pVIc, when added to Hep-2 cells infected with adenovirus serotype 5, inhibited th e synthesis of infectious virus, presumably by prematurely activating the p roteinase so that it cleaved virion precursor proteins before virion assemb ly, thereby aborting the infection.