Site-directed mutagenesis probing the catalytic role of arginines 165 and 166 of human cytomegalovirus protease

Biochemistry
P H LiangS S Abdel-Meguid

Abstract

Human cytomegalovirus (CMV) is a member of the Herpesviridae family of viruses that also includes herpes simplex viruses (HSV-1 and HSV-2), varicella-zoster virus (VZV), human herpes virus-6, 7, and 8 (HHV-6, HHV-7, and HHV-8), and Epstein-Barr virus (EBV). Each member of this family encodes a serine protease that is a potential target for antiviral therapeutic intervention. We recently reported the crystal structure of CMV proteases [Qiu, X., Culp, J. S., DiLella, A. G., Hellmig, B., Hoog, S. S., Janson, C. A., Smith, W. W., and Abdel-Meguid, S. S. (1996) Nature 383, 275-279] and proposed that the highly conserved Arg165 and Arg166 residues are involved in stabilizing the oxyanion intermediate in human herpes protease catalyzed reactions through the backbone NH and side chain, respectively. In the current study, site-directed mutagenesis was carried out to probe the catalytic function of these two amino acid residues. Substitution of Arg166 with an alanine has led to ablation of enzymatic activity without detectable change in CMV protease conformation, supporting suggestions from the crystal structure that Arg166 side chain plays a major role in catalysis. The wild-type has a Km = 138 +/- 17 microM and kcat = 19.9 +/- 1.1 min-...Continue Reading

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