IN-VITRO PROTEOLYTIC ACTIVITY AND ACTIVE-SITE IDENTIFICATION OF THE HUMAN CYTOMEGALOVIRUS PROTEASE

Human cytomegalovirus (HCMV) encodes a protease that cleaves itself an d the HCMV assembly protein. Two proteolytic processing sites within t he protease were identified at Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site). Identification of rP5-P4' and mP4-P6' a s the minimal peptide substrates spanning the release and maturation c leavage sites, respectively, demonstrated a requirement for residues f lanking the conserved core in substrate recognition and hydrolysis, wh ich are unique to HCMV. Kinetic parameters determined for release-site -derived and maturation-site-derived peptides revealed a 10-fold incre ase in k(cat)/K-m for a maturational peptide (mP4-P8') over release-si te peptide (rP5-P5'). Experimental results with a panel of class-speci fic protease inhibitors were consistent with the protease being a memb er of the serine or cysteine family of proteases. Further investigatio n revealed that the HCMV protease activity decreased with incorporatio n of [C-14]iodoacetic acid, but when approximately 4.5 mol C-14 were i ncorporated/mel enzyme, the enzyme retained approximately 20% of its o riginal activity, indicating that hydrolysis does not require a cystei ne nucleophile. Analysis of diisopropyl-fluorophosphate-inactivated pr otease by mass spectrometry indicated a stoichiometry of 1 diisopropyl phosphate/protease molecule, suggesting that hydrolysis requires a si ngle serine nucleophile. The residue modified by diisopropyl fluoropho sphate was identified as Ser132 by modification with H-3-labeled diiso propyl fluorophosphate, peptide mapping and Edman degradation. This re sidue and the region in which it is found is highly conserved among th e herpes virus proteases. These data demonstrates that HCMV protease i s a serine protease and that Ser132 is the active-site nucleophile.