HUMAN CYTOMEGALOVIRUS PROTEINASE - CANDIDATE GLUTAMIC-ACID IDENTIFIEDAS 3RD MEMBER OF PUTATIVE ACTIVE-SITE TRIAD

The human cytomegalovirus (HCMV) proteinase is synthesized as a 709-am ino-acid precursor that undergoes at least three autoproteolytic cleav ages. 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 viru ses contain five highly conserved domains (CD1 through CD5). An absolu tely conserved serine in CD3 has been shown by site-directed mutagenes is 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 absolu tely conserved histidine in CD2 has also been demonstrated by site-dir ected mutagenesis of the SCMV and HSV-1 enzymes to be essential for pr oteolytic activity and has been proposed to be a second member of the catalytic triad of this serine proteinase. We report here the use of s ite-directed mutagenesis to investigate the active-site amino acids of HCMV assemblin. Substitutions were made for the CD3 serine and CD2 hi stidine residues implicated as active-site components, and for other a mino acids whose influence on enzyme activity was of interest. The mut ant proteinases were tested in a transient transfection assay for thei r ability to cleave their natural substrate, the assembly protein prec ursor. Results of these experiments verified that HCMV CD3 serine (Ser -132) and CD2 histidine (His-63) are essential for proteolytic activit y and identified a glutamic acid (Glu-122) within CD3 that is also ess ential for proteolytic activity and may be conserved among all herpesv irus assemblin homologs. We suggest that CD3 Glu-122, CD3 Ser-132, and CD2 His-63 constitute the active-site triad of this serine proteinase .