Proton transfer in the mechanism of polyadenylate polymerase.
Proton transfer in the mechanism of polyadenylate polymerase.
复制标题
质子转移在聚腺苷酸聚合酶的机理中。
DOI：
10.1042/bj20082019
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发表时间：
2009-05-13
期刊：
影响因子：
--
通讯作者：
Bohm A
中科院分区：
文献类型：
--
作者：
Balbo PB;Bohm A
Polyadenylate polymerase (PAP) 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, the pH dependence of the steady state kinetic parameters of yeast PAP were determined for the forward (adenylyltransfer) and reverse (pyrophosphorolysis) reactions. The results indicate that productive formation of an enzyme-RNA-MgATP complex is pH independent over a broad pH range, but that formation of an active enzyme-RNA-MgPPi complex is strongly pH dependent, consistent with the production of a proton on the enzyme in the 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 be protonated, and the other, deprotonated. The deuterium solvent isotope effect on Vmax is also consistent with proton transfer involved in catalysis of a rate determining step. Finally, pKa calculations of PAP were performed by the multiconformational continuum electrostatic (MCCE) method. Together, the data support that the protonation of residues K215 and Y224 exhibit cooperativity important for MgATP2− and MgPPi2− binding/dissociation, and suggest these residues function in electrostatic, but not in general acid catalysis.
