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

The interaction of the human adenovirus proteinase (AVP) and AVP-DNA complexes with the 11-amino acid cofactor pVIc was characterized. The equilibrium dissociation constant for the binding of pVIc to AVP was 4.4 microM. The binding of AVP to 12-mer single-stranded DNA decreased the K(d) for the binding of pVIc to AVP to 0.09 microM. The pVIc-AVP complex hydrolyzed the substrate with a Michaelis constant (K(m)) of 3.7 microM and a catalytic rate constant (k(cat)) of 1.1 s(-1). In the presence of DNA, the K(m) increased less than 2-fold, and the k(cat) increased 3-fold. Alanine-scanning mutagenesis was performed to determine the contribution of individual pVIc side chains in the binding and stimulation of AVP. Two amino 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 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 cofactors, pVIc or the viral DNA, to AVP did not dramatically alter its secondary structure as determined by vacuum ultraviolet circular dichroism. pVIc, when added to Hep-2 cells infected with adenovirus serotype 5, inhibited the synthesis of infectious virus, presumably by prematurely activating the proteinase so that it cleaved virion precursor proteins before virion assembly, thereby aborting the infection.

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 protein pVI (pVIc) and the viral DNA. DNA binding was monitored by changes in enzyme activity or by fluorescence anisotropy. The equilibrium dissociation constants for the binding of AVP and AVP-pVIc complexes to 12-mer double-stranded (ds) DNA were 63 and 2.9 nM, respectively. DNA binding was not sequence specific; the stoichiometry of binding was proportional to the length of the DNA. Three molecules of the AVP-pVIc complex bound to 18-mer dsDNA and six molecules to 36-mer dsDNA. When AVP-pVIc complexes bound to 12-mer dsDNA, two sodium ions were displaced from the DNA. A Delta of -4.6 kcal for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy results from nonspecific interactions between the AVP-pVIc complex and DNA. The cofactors altered the interaction of the enzyme with the fluorogenic substrate (Leu-Arg-Gly-Gly-NH)2-rhodamine. In the absence of any cofactor, the Km was 94.8 microM and the kcat was 0.002 s(-1). In the presence of adenovirus DNA, the Km decreased 10-fold and the kcat increased 11-fold. In the presence of pVIc, the Km decreased 10-fold and the kcat increased 118-fold. With both cofactors present, the kcat/Km ratio increased 34000-fold, compared to that with AVP alone. Binding to DNA was coincident with stimulation of proteinase activity by DNA. Although other proteinases have been shown to bind to DNA, stimulation of proteinase activity by DNA is unprecedented. A model is presented suggesting that AVP moves along the viral DNA looking for precursor protein cleavage sites much like RNA polymerase moves along DNA looking for a promoter.

Temporal and spatial control of the adenovirus proteinase by both a peptide and the viral DNA.

The adenovirus proteinase (AVP) uses both an 11-amino acid peptide (pVIc) and the viral DNA as cofactors to increase its catalytic rate constant 6000-fold. The crystal structure of an AVP-pVIc complex at 2.6-A resolution reveals a new protein fold of an enzyme that is the first member of a new class of cysteine proteinases, which arose via convergent evolution.

Different modes of inhibition of human adenovirus proteinase, probably a cysteine proteinase, by bovine pancreatic trypsin inhibitor.

The type of proteinase and the nature of the active site of the human adenovirus proteinase are unknown. For these reasons we produced an inhibitor profile of the enzyme. Enzyme activity in disrupted virions was inhibited by several serine-specific as well as cysteine-specific proteinase inhibitors. Of the inhibitors that worked, the most useful potentially in illuminating the nature of the active site was bovine pancreatic trypsin inhibitor (BPTI), and for this reason we extensively characterized the interaction with BPTI. In disrupted virions, the enzyme is irreversibly inhibited by BPTI with a Ki of 35 nM and a ki of 6.2 x 10(-4) s(-1). One reason enzyme activity is inhibited is that BPTI, a basic protein, precipitates the viral DNA, a cofactor of enzyme activity. In vitro with purified components, BPTI acts as a competitive inhibitor (Ki 2 microM) of the recombinant proteinase complexed with its 11-amino-acid cofactor pVIc. The recombinant endoproteinase is beat labile whereas its 11-amino-acid cofactor is heat stable. We estimate there are about 50 molecules of proteinase per virus particle.

Characterization of human adenovirus proteinase activity in disrupted virus particles.

Virus-coded proteinases are attractive targets for antiviral therapy; however, lack of quick, sensitive, quantitative, and selective assays for enzyme activity makes it difficult to characterize these proteinases and to screen large numbers of potential inhibitors. Here we describe new substrates for the adenovirus proteinase, fluorogenic Rhodamine-based substrates containing tetrapeptides corresponding to sequences cleaved in adenovirus precursor proteins. Proteinase activity in as few as 10(9) disrupted virions could be quantitatively detected in a 30-min assay. With the substrate (Leu-Arg-Gly-Gly-NH)2-Rhodamine, the Km was 1.4 microM and the Vmax was 3.24 pmol substrate hydrolyzed/sec/pmol virus. Enzyme activity was stimulated by dithiothreitol and inhibited by several serine-specific as well as cysteine-specific proteinase inhibitors. In a thiol protection experiment, the virion enzyme was shown to have a cysteine residue with an unusually low pKa, a pKa similar to that of the active-site nucleophile of the cysteine proteinase papain. The curve of Vmax as a function of pH is unlike the curve from papain and implied that there are at least three ionizable groups whose protonation state can affect catalysis - one with a pKa of 6.2, another with a pKa of 7.2, and a third with a pKa of 8.3.

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 Km, which changes by no more than 2-fold, but by increasing kcat. rEP alone had a small amount of activity, the kcat of which increased 355-fold with pVIc and 6072-fold with adenovirus serotype 2 (Ad2) DNA as well. Curves of Vmax of rEP.pVIc complexes with the substrate (Leu-Arg-Gly-NH)2-rhodamine as a function of pH in the absence and presence of Ad2 DNA indicate that the pKa values of amino acids that affect catalysis are quite different from those that affect catalysis by the cysteine proteinase papain. The pKa 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.

Viral DNA and a viral peptide can act as cofactors of adenovirus virion proteinase activity.

Human adenovirus (Ad2), like many other viruses, contains a virion-associated proteinase essential for the synthesis of infectious virus particles. We observed proteinase activity in wild-type virus but not in the ts-1 virus, which contains a mutation in the Ad2 L3 endoprotease gene that confers temperature-sensitive processing of virion precursor proteins. Unexpectedly, we did not observe proteinase activity with purified recombinant endoprotease protein (M(r) 23 K). Purified recombinant endoprotease protein, however, complemented the mutation in ts-1 virions, restoring proteinase activity when mixed together. This implied that cofactors may be required. Here we reconstitute proteinase activity in vitro with three purified viral components: (1) the recombinant endoprotease protein; (2) an 11-amino-acid peptide that originates from the carboxy terminus of pVI, the precursor to virion component VI; and (3) adenovirus DNA. The use of DNA for a proteinase activity is unprecedented.

Structure of an acyl-enzyme intermediate during catalysis: (guanidinobenzoyl)trypsin.

The crystal and molecular structure of trypsin at a transiently stable intermediate step during catalysis has been determined by X-ray diffraction methods. Bovine trypsin cleaved the substrate p-nitrophenyl p-guanidinobenzoate during crystallization under conditions in which the acyl-enzyme intermediate, (guanidinobenzoyl)trypsin, was stable. Orthorhombic crystals formed in space group P2(1)2(1)2(1), with a = 63.74, b = 63.54, and c = 68.93 A. This is a crystal form of bovine trypsin for which a molecular structure has not been reported. Diffraction data were measured with a FAST (Enraf Nonius) diffractometer. The structure was refined to a crystallographic residual of R = 0.16 for data in the resolution range 7.0-2.0 A. The refined model of (guanidinobenzoyl)trypsin provides insight into the structural basis for its slow rate of deacylation, which in solution at 25 degrees C and pH 7.4 exhibits a t1/2 of 12 h. In addition to the rotation of the Ser-195 hydroxyl away from His-157, C beta of Ser-195 moves 0.7 A toward Asp-189 at the bottom of the active site, with respect to the native structure. This allows formation of energetically favorable H bonds and an ion pair between the carboxylate of Asp-189 and the guanidino group of the substrate. This movement is dictated by the rigidity of the aromatic ring in guanidinobenzoate--model-building indicates that this should not occur when arginine, with its more flexible aliphatic backbone, forms the ester bond with Ser-195. As a consequence, highly ordered water molecules in the active site are no longer close enough to the scissile ester bond to serve as potential nucleophiles for hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)