Kinetics of the dimerization of retroviral proteases: the "fireman's grip" and dimerization |
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Authors: | Ingr Marek Uhlíková Tat'ána Strísovský Kvido Majerová Eva Konvalinka Jan |
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Affiliation: | Institute of Organic Chemistry and Biochemistry, Academy of Science of the Czech Republic, 166 10 Praha 6, Czech Republic. |
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Abstract: | All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the "fireman's grip," which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp-Thr(Ser)-Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp-Thr-Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild-type and mutant proteases from HIV and myeloblastosis-associated virus harboring either Ser or Thr in their Asp-Thr(Ser)-Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be approximately 10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation. |
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Keywords: | Retroviral protease dimerization HIV protease fireman's grip kinetic assay fluorescence pressure HIV‐1‐PR(T), wild‐type HIV‐1‐PR HIV‐1‐PR(S), HIV‐1‐PR harboring Thr26Ser mutation MAV‐PR(S), wild‐type MAV‐PR MAV‐PR(T), MAV‐PR harboring Ser38Thr mutation MPMV‐PR(T), wild‐type MPMV‐PR, 12‐kD form DABCYL, 4‐[[4′‐(dimethylamino)phenyl]azo]‐benzoic acid EDANS, 5‐[(2′aminoethyl)‐amino]naphtalenesulfonic acid MAV, myeloblastosis‐associated virus MPMV, Mason‐Pfizer monkey virus PAL, peptide amide linker, 5‐(4‐Fmoc‐aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid PheSta, phenylstatin PR, protease RSV, Rous sarcoma virus TBTU, O‐(benzotriazol‐1‐yl)‐N,N,N′,N′‐tetramethyluronium tetrafluoroborate Fmoc, 9‐fluorenylmethoxycarbonyl TFA, trifluoroacetic acid PMSF, phenylmethylsulfonyl fluoride |
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