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[摘要]：PAP (polyadenylate polymerase) is the template-independent RNA polymerase responsible for synthesis of the 3' poly(A) tails of mRNA. To investigate the role of proton transfer in the catalytic mechanism of PAP, file pH dependence of the steady-slate kinetic parameters of yeast PAP were determined for the forward (adenyl transfer) and reverse (pyrophosphorolysis) reactions. The results indicate that productive formation of an enzyme-RNA-MaATP complex is pH independent over it broad pH range, but that formation of an active enzyme-RNA-M-PP, complex is strongly pH dependent, consistent with the production of a proton oil the enzyme in file forward reaction. The pH dependence of the maximum velocity of the forward reaction suggests two protonic species are involved in enzyme catalysis. Optimal enzyme activity requires one species to he protonated and the other deprotonated. The deuterium solvent isotope effect oil V,: is also consistent with proton transfer involved in catalysis of it rate-determining, step. Finally, pK(a) calculations of PAP were performed by the MCCE (multiconformational continuum electrostatic) method. Together, the data support that the protonation of residues Lys(215) and Tyr(224) exhibit co-operativity that is important for MgATP(2) zinc MgPPi2- binding/dissociation, and suggest these residues function in electrostatic. but not in general acid. catalysis. |
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