| Abstract
| - The interaction of the human adenovirus proteinase (AVP) and AVP−DNA complexes withthe 11-amino acid cofactor pVIc was characterized. The equilibrium dissociation constant for the bindingof pVIc to AVP was 4.4 μM. The binding of AVP to 12-mer single-stranded DNA decreased the Kd forthe binding of pVIc to AVP to 0.09 μM. The pVIc−AVP complex hydrolyzed the substrate with a Michaelisconstant (Km) of 3.7 μM and a catalytic rate constant (kcat) of 1.1 s-1. In the presence of DNA, the Kmincreased less than 2-fold, and the kcat increased 3-fold. Alanine-scanning mutagenesis was performed todetermine the contribution of individual pVIc side chains in the binding and stimulation of AVP. Twoamino acid residues, Gly1‘ and Phe11‘, were the major determinants in the binding of pVIc to AVP,while Val2‘ and Phe11‘ were the major determinants in stimulating enzyme activity. Binding of AVP toDNA greatly suppressed the effects of the alanine substitutions on the binding of mutant pVIcs to AVP.Binding of either or both of the cofactors, pVIc or the viral DNA, to AVP did not dramatically alter itssecondary structure as determined by vacuum ultraviolet circular dichroism. pVIc, when added to Hep-2cells infected with adenovirus serotype 5, inhibited the synthesis of infectious virus, presumably byprematurely activating the proteinase so that it cleaved virion precursor proteins before virion assembly,thereby aborting the infection.
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