Characterization of the monomer-dimer equilibrium of human cytomegalovirus protease by kinetic methods

Herpesviruses encode a serine protease that is essential for the maturation of infectious virions. This protease has a unique polypeptide backbone fold and contains a novel Ser-His-His catalytic triad. It exists in a monomer-dimer equilibrium in solution, but only the dimer form of the enzyme is catalytically active. The stability of this dimer is affected by the presence of anti-chaotropic agents. Most of the reported K-d values for this dimer (between 0.6 and 6 muM) are inconsistent with the fact that the protease is routinely assayed at 20-50 nM concentrations, as only monomeric species would be expected with such K-d values. We have characterized the monomer-dimer equilibrium of HCMV protease using a new method, which observes the exchange between dimers of the wild-type enzyme and the active-site Ser132A1a mutant in a titration experiment. The K-d of the dimer was determined to be 8 muM and 31 nM in the absence or presence of anti-chaotropic agents (10% glycerol and 0.5 M Na2SO4), respectively. Detailed kinetic analysis also showed that, in addition to the 260-fold stabilization of the dimer, the anti-chaotropic agents produced a 7-fold enhancement in the catalytic activity of the dimer.