RESUMO
The single-chain 28 kDa human cytomegalovirus (HCMV) protease catalytic domain containing the A143Q mutation has been kinetically and conformationally characterized. The specific activity of the HCMV A143Q protease (HCMVp) increases as the protease concentration increases, suggesting that this protease oligomerizes at high protein concentration to form a more active species. Both cross-linking and light-scattering studies of HCMVp show the existence of a homodimer with an apparent molecular mass of 56 kDa under low ionic strength and high protein concentration. The cosolvent and solute effects of glycerol, trisodium citrate, and NaCl as well as the temperature effects on the HCMVp activity and quaternary structure were investigated. The effects induced by cosolvents and temperature can largely be explained by their influences in the dimerization or oligomerization state of HCMVp. The dissociation constant (Kd) for the HCMVp homodimer was determined to be 8 +/- 1 microM with all activity attributed to the dimeric form. Monomeric HCMVp is inactive. This report demonstrates that in vitro, HCMV A143Q protease exists as an obligate catalytic homodimer. This protease dimerization may have regulatory significance during viral replication.
Assuntos
Citomegalovirus/enzimologia , Endopeptidases/metabolismo , Serina Endopeptidases , Catálise , Citratos/farmacologia , Ácido Cítrico , Reagentes de Ligações Cruzadas , Endopeptidases/química , Endopeptidases/efeitos dos fármacos , Endopeptidases/genética , Transferência de Energia , Estabilidade Enzimática , Escherichia coli/genética , Glicerol/farmacologia , Temperatura Alta , Cinética , Luz , Modelos Químicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Espalhamento de Radiação , Cloreto de Sódio/farmacologiaRESUMO
Proteolytic processing of capsid assembly protein precursors by herpesvirus proteases is essential for virion maturation. A 2.5 A crystal structure of the human cytomegalovirus protease catalytic domain has been determined by X-ray diffraction. The structure defines a new class of serine protease with respect to global-fold topology and has a catalytic triad consisting of Ser-132, His-63, and His-157 in contrast with the Ser-His-Asp triads found in other serine proteases. However, catalytic machinery for activating the serine nucleophile and stabilizing a tetrahedral transition state is oriented similarly to that for members of the trypsin-like and subtilisin-like serine protease families. Formation of the active dimer is mediated primarily by burying a helix of one protomer into a deep cleft in the protein surface of the other.