The effect of internal autocleavage on 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 inactivate the protease and provide a mechanism for the negative regulation of enzyme activity during viral infection. We purified recombinant enzymes to demonstrate I site autocleavage in vitro and used site-directed mutagenesis of the I site to stabilize the protease. No difference in the kinetic properties of wild type and stabilized mutant proteases were observed in an in vitro peptide cleavage assay. The consequences of I site cleavage on enzyme activity were investigated two ways. First, autodigestion of the wild type enzyme converted the intact protease to N15 and C13 autocleavage products without a corresponding loss in enzyme activity. Second, genetic constructs encoding the N15 and C13 autocleavage products were generated and expressed separately in Escherichia coli, and each fragment was purified. An active enzyme was reconstituted 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 optimal reaction conditions. We concluded from these studies that I site cleavage does not inactivate the HCMV protease, in the absence of other virally induced factors, and that limited potential exists for the regulation of catalytic activity by I site cleavage.
O'Boyle
D R
DR
Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.
Wager-Smith
K
K
Stevens
J T
JT
Weinheimer
S P
SP
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002461:Cell Line, Transformed; D003587:Cytomegalovirus / Q000201:enzymology*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides
D009838:Oligodeoxyribonucleotides; D010450:Endopeptidases