SITE-DIRECTED MUTAGENESIS PROBING THE CATALYTIC ROLE OF ARGININE-165 AND ARGININE-166 OF HUMAN CYTOMEGALOVIRUS PROTEASE

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, H HV-7, and HHV-8), and Epstein-Barr virus (EBV). Each member of this fa mily encodes a serine protease that is a potential target for antivira l 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. (199 6) Nature 383, 275-279] and proposed that the highly conserved Arg165 and Arg166 residues are involved in stabilizing the oxyanion intermedi ate in human herpes protease catalyzed reactions through the backbone NH and side chain, respectively; In the current study, site-directed m utagenesis was carried out to probe the catalytic function of these tw o amino acid residues. Substitution of Arg166 with an alanine has led to ablation of enzymatic activity without detectable change in CMV pro tease conformation, supporting suggestions from the crystal structure that Arg166 side chain plays a major role in catalysis. The wild-type has a K-m = 138 +/- 17 mu M and k(cat) = 19.9 +/- 1.1 min(-1), while R 166A has only residual activity, with a k(cat) = 0.012 +/- 0.001 min(- 1) and an unaltered K-m = 145 +/- 18 mu M. In the crystal structure, t he side chain of Arg166 was shown previously to hold a water molecule that can act as a hydrogen-bond donor to the oxyanion and was thus pro posed to stabilize the oxyanion intermediate. However, kinetic charact erization of the mutant R165A only reveals a 2.7-fold lower activity t han wild-type, with a K-m = 166 +/- 19 mu M and a k(cat) = 7.4 +/- 0.4 min(-1). These results confirm that Arg165 side chain is not involved in the stabilization of the oxyanion. It is likely that Arg165 only u tilizes the backbone NH for catalysis as suggested by the crystal stru cture.