Mutagenesis of conserved residues of the adenovirus protease.

Based on the alignment of 12 adenovirus protease sequences, we have identified eleven conserved residues for mutagenesis. Eight of these, E5, D26, N44, E71, D77, D102, N144, and N170, are potential candidates for the third residue of the active site triad. N44, E71, N144, and N170 proved to be essential for enzyme activity. Glutamic acid 71 was proposed for the active site. Mutation of the three conserved cysteines suggested that C122 is the active nucleophile, C104 is the target for activation by peptide pVIc, and C126 is dispensable. Rescue of enzyme activity of the C104 G mutant by pVIc suggested that disulfide bond formation between the peptide and the protease may not be absolutely essential for stimulation of enzyme activity.