THE EFFECT OF INTERNAL AUTOCLEAVAGE OIL KINETIC-PROPERTIES OF THE HUMAN CYTOMEGALOVIRUS PROTEASE CATALYTIC DOMAIN

The 28-kilodalton (kDa) catalytic domain of the human cytomegalovirus (HCMV) protease undergoes autoproteolytic cleavage at an internal site (I site), yielding amino terminal 15-kDa (N15) and carboxyl terminal 13-kDa (C13) fragments. I site autocleavage has been postulated to ina ctivate the protease and provide a mechanism for the negative regulati on of enzyme activity during viral infection, We purified recombinant enzymes to demonstrate I site autocleavage in vitro and used site dire cted mutagenesis of the I site to stabilize the protease. No differenc e in the kinetic properties of wild type and stabilized mutant proteas es were ob served in an in vitro peptide cleavage assay. The consequen ces of I site cleavage on enzyme activity were investigated two ways. First, autodigestion of the wild type enzyme converted the intact prot ease to N15 and C13 autocleavage products without a corresponding loss in enzyme activity. Second, genetic constructs encoding the N15 and C 13 autocleavage products were generated and expressed separately in Es cherichia coil, and each fragment was purified. An active enzyme was r econstituted by refolding a mixture of the purified fragments in vitro to form a noncovalent complex. The kinetic properties of this complex were very similar to the wild type and stabilized enzymes under optim al reaction conditions. We concluded from these studies that I site cl eavage does not inactivate the HCMV protease, in the absence of other virally induced factors, and that limited potential exists for the reg ulation of catalytic activity by I site cleavage.