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 Kd values for this dimer (between 0.6 and 6 microM) 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 Kd 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 Ser132Ala mutant in a titration experiment. The Kd of the dimer was determined to be 8 microM 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.
Khayat
Reza
R
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Batra
Renu
R
Bebernitz
Geraldine A
GA
Olson
Matthew W
MW
Tong
Liang
L
D002021:Buffers; D002384:Catalysis; D003587:Cytomegalovirus / Q000201:enzymology* / Q000235:genetics; D019281:Dimerization; D004229:Dithiothreitol / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009025:Morpholines / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics; D013431:Sulfates / Q000737:chemistry; D013499:Surface Properties; D017186:Titrimetry
D002021:Buffers; D009025:Morpholines; D013431:Sulfates; C012036:sodium sulfate; C008550:morpholinopropane sulfonic acid; D012965:Sodium Chloride; D004492:Edetic Acid; D012697:Serine Endopeptidases; D004229:Dithiothreitol