Human cytomegalovirus proteinase: candidate glutamic acid identified as third member of putative active-site triad.
The human cytomegalovirus (HCMV) proteinase is synthesized as a 709-amino-acid precursor that undergoes at least three autoproteolytic cleavages. The mature proteinase, called assemblin, is one of the products of autoproteolysis and is composed of the first 256 amino acids of the precursor. HCMV assemblin and its homologs in other herpes group viruses contain five highly conserved domains (CD1 through CD5). An absolutely conserved serine in CD3 has been shown by site-directed mutagenesis of the simian cytomegalovirus (SCMV) and herpes simplex virus type 1 (HSV-1) enzymes and by inhibitor affinity labeling of the HSV-1 and HCMV enzymes to be the active-site nucleophile of assemblin. An absolutely conserved histidine in CD2 has also been demonstrated by site-directed mutagenesis of the SCMV and HSV-1 enzymes to be essential for proteolytic activity and has been proposed to be a second member of the catalytic triad of this serine proteinase. We report here the use of site-directed mutagenesis to investigate the active-site amino acids of HCMV assemblin. Substitutions were made for the CD3 serine and CD2 histidine residues implicated as active-site components, and for other amino acids whose influence on enzyme activity was of interest. The mutant proteinases were tested in a transient transfection assay for their ability to cleave their natural substrate, the assembly protein precursor. Results of these experiments verified that HCMV CD3 serine (Ser-132) and CD2 histidine (His-63) are essential for proteolytic activity and identified a glutamic acid (Glu-122) within CD3 that is also essential for proteolytic activity and may be conserved among all herpesvirus assemblin homologs. We suggest that CD3 Glu-122, CD3 Ser-132, and CD2 His-63 constitute the active-site triad of this serine proteinase.
Cox
G A
GA
Infectious Diseases Research Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Wakulchik
M
M
Sassmannshausen
L M
LM
Gibson
W
W
Villarreal
E C
EC
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000032:analysis; D001665:Binding Sites; D003587:Cytomegalovirus / Q000201:enzymology*; D010450:Endopeptidases / Q000737:chemistry*; D018698:Glutamic Acid / Q000032:analysis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012697:Serine Endopeptidases
D001224:Aspartic Acid; D018698:Glutamic Acid; D010450:Endopeptidases; D012697:Serine Endopeptidases; C071910:assemblin