In vitro proteolytic activity and active-site identification of the human cytomegalovirus protease.

Human cytomegalovirus (HCMV) encodes a protease that cleaves itself and the HCMV assembly protein. Two proteolytic processing sites within the protease were identified at Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site). Identification of rP5-P4' and mP4-P6' as the minimal peptide substrates spanning the release and maturation cleavage sites, respectively, demonstrated a requirement for residues flanking the conserved core in substrate recognition and hydrolysis, which are unique to HCMV. Kinetic parameters determined for release-site-derived and maturation-site-derived peptides revealed a 10-fold increase in kcat/Km for a maturational peptide (mP4-P8') over release-site peptide (rP5-P5'). Experimental results with a panel of class-specific protease inhibitors were consistent with the protease being a member of the serine or cysteine family of proteases. Further investigation revealed that the HCMV protease activity decreased with incorporation of [14C]iodoacetic acid, but when approximately 4.5 mol 14C were incorporated/mol enzyme, the enzyme retained approximately 20% of its original activity, indicating that hydrolysis does not require a cysteine nucleophile. Analysis of diisopropyl-fluorophosphate-inactivated protease by mass spectrometry indicated a stoichiometry of 1 diisopropyl phosphate/protease molecule, suggesting that hydrolysis requires a single serine nucleophile. The residue modified by diisopropyl fluorophosphate was identified as Ser132 by modification with 3H-labeled diisopropyl fluorophosphate, peptide mapping and Edman degradation. This residue and the region in which it is found is highly conserved among the herpes virus proteases. These data demonstrates that HCMV protease is a serine protease and that Ser132 is the active-site nucleophile.