Investigating the role of histidine 157 in the catalytic activity of humancytomegalovirus protease

Herpesvirus proteases belong to a new class of serine proteases and contain a novel Ser-His-His catalytic triad, while classical serine proteases have an acidic residue as the third member. To gain a better understanding of t he molecular basis for the functional role of the third-member His residue, we have carried out structural and biochemical investigations of human cyt omegalovirus (HCMV) protease that bears mutations of the His157 third membe r. Kinetic studies showed that all the mutants have reduced catalytic activ ity. Structural studies revealed that a solvent molecule is hydrogen-bonded to the His63 second member and Ser134 in the H157A mutant, partly rescuing the activity of this mutant. This is confirmed by our kinetic and structur al observations on the S134A/H157A double mutant, which showed further redu ctions in the catalytic activity. The structure of the H157A mutant is also in complex with the PMSF inhibitor. The H157E mutant has the best catalyti c activity among the mutants; its structure, however, showed conformational readjustments of the His63 and Ser132 residues. The Ser132-His63 diad of H CMV protease has similar activity as the diads in classical serine protease s, whereas the contribution of the His157 third member to the catalysis is much smaller. Finally, structural comparisons revealed the presence of two conserved structural water molecules at the bottom of the S-1 pocket, sugge sting a possible new direction for the design of HCMV protease inhibitors.