Human adenovirus proteinase: DNA binding and stimulation of proteinase activity by DNA

The interaction of the human adenovirus proteinase (AVP) with various DNAs was characterized. AVP requires two cofactors for maximal activity, the 11- amino acid residue peptide from the C-terminus of adenovirus precursor prot ein pVI (pVIc) and the viral DNA. DNA binding was monitored by changes in e nzyme activity or by fluorescence anisotropy. The equilibrium dissociation constants for the binding of AVP and AVP-pVIc complexes to 12-mer double-st randed (ds) DNA were 63 and 2.9 nM, respectively. DNA binding was not seque nce specific; the stoichiometry of binding was proportional to the length o f the DNA. Three molecules of the AVP-pVIc complex bound to 18-mer dsDNA an d six molecules to 36-mer dsDNA. When AVP-pVIc complexes bound to 12-mer ds DNA, two sodium ions were displaced from the DNA. A DeltaG(0)(0) of -4.6 kc al for the nonelectrostatic free energy of binding indicated that a substan tial component of the binding free energy results from nonspecific interact ions between the AVP-pVIc complex and DNA. The cofactors altered the intera ction of the enzyme with the fluorogenic substrate (Leu-Arg-Gly-Gly-NH)(2)- rhodamine. In the absence of any cofactor, the K-m was 94.8 muM and the kca l was 0.002 s(-1). In the presence of adenovirus DNA, the Km decreased 10-f old and the k(cat) increased 11-fold. In the presence of pVIc, the K-m decr eased 10-fold and the k(cat) increased 118-fold. With both cofactors presen t, the k(cat)/K-m ratio increased 34000-fold, compared to that with AVP alo ne. Binding to DNA was coincident with stimulation of proteinase activity b y DNA. Although other proteinases have been shown to bind to DNA, stimulati on of proteinase activity by DNA is unprecedented. A model is presented sug gesting that AVP moves along the viral DNA looking for precursor protein cl eavage sites much like RNA polymerase moves along DNA looking for a promote r.