Adenovirus Type 2 Endoprotease: Isoforms and Redox Effects

Abstract

The cysteine protease encoded by adenovirus type 2 contains eight cysteines, some of which are involved in catalysis and enzyme activation. Here we investigated the effects of oxidation, mercaptoethanol, dithiothreitol, diamide and protein disulphide isomerase on wild-type and mutant enzymes. Three isoforms of the enzyme were detected in infected cells and a fourth in preparations of purified recombinant enzyme. The latter isoform was absent in preparations of enzyme mutated at any of the three conserved cysteines, C-104, C-122 and C-126. Enzyme activity could be stimulated by agents other than the authentic activating peptide (pVIc), such as cysteamine, though less efficiently. Diamide at low concentrations stimulated the activity of the tsl enzyme, but inhibited both tsl and wild-type enzyme at higher concentrations. Protein disulphide isomerase failed to restore enzyme activity to the oxidized isoform. The present studies in combination with previous results using mutants appeared to rule out amino acids C-67, C-122, C-126 and C-127, leaving the two remaining semi-conserved C-17 and C-40 and the conserved C-104 as potential candidates for binding peptide pVIc.

Viral Proteases

“…The Val2 side chain is completely buried in the complex, and modifications near the N-terminus of the peptide have deleterious effects on the binding and activation. , A disulfide bond is observed between Cys10 of the peptide and Cys104 of the enzyme (Figure k), the other strictly conserved Cys residues among adenovirus proteases. Biochemical data also suggested the requirement of this disulfide bond. ,, A recent report showed however that the presence of DNA greatly suppresses the functional importance of this disulfide …”

“…Biochemical data also suggested the requirement of this disulfide bond. 238,241,252 A recent report showed however that the presence of DNA greatly suppresses the functional importance of this disulfide. 253 However, the pVIc peptide is located far from the active site of the enzyme, suggesting an indirect mechanism in the protease activation by this peptide.…”