Kinetic studies of yeast polyA polymerase indicate an induced fit mechanism for nucleotide specificity
Kinetic studies of yeast polyA polymerase indicate an induced fit mechanism for nucleotide specificity
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DOI：
10.1021/bi050089r
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发表时间：
2005-05-31
期刊：
影响因子：
2.9
通讯作者：
Bohm, A
中科院分区：
文献类型：
--
作者：
Balbo, PB;Meinke, G;Bohm, A
多聚腺苷聚合酶(PAP)催化3‘-多聚腺苷酸尾的合成到mRNA上。对PAP进行了全面的稳态动力学分析，包括正向和反向反应的初始速度研究、抑制研究和替代底物的使用。反应(A(N)+ATPA(n+1)+PPI)可用快速平衡随机机理描述。测定或计算了反应的几个热力学参数，包括总平衡常数(K-eq=84)和内部步骤的表观平衡常数(K-int=4)，这涉及到中心络合物的速率决定相互转化。V-max的巨大差异(100倍)解释了核苷酸特异性(ATP与CTP)，尽管K-m只有3倍的差异。比较硫元素对ATP和CTP的V-max的影响，表明这两个反应的化学步骤都是速率决定的。比较硫元素对V-max/K-m的影响，揭示了两种核苷酸结合的不同机制。与这些数据一致，提出了一种核苷酸特异性的诱导匹配机制，即PAP将选择ATP的统一结合机制与加速正确底物的速度的基态失稳机制结合在一起。
Polyadenylate polymerase (PAP) catalyzes the synthesis of 3'-polyadenylate tails onto mRNA. A comprehensive steady-state kinetic analysis of PAP was conducted which included initial velocity studies of the forward and reverse reactions, inhibition studies, and the use of alternative substrates. The reaction (A(n) + ATP A(n+1) + PPi) is adequately described by a rapid equilibrium random mechanism. Several thermodynamic parameters for the reaction were determined or calculated, including the overall equilibrium constant (K-eq = 84) and the apparent equilibrium constant of the internal step (K-int = 4) which involves the rate-determining interconversion of central complexes. A large (100-fold) difference in V-max accounts for nucleotide specificity (ATP vs CTP), despite an only 3-fold difference in K-m. Comparison of the sulfur elemental effect on V-max for ATP and CTP suggests that the chemical step is rate-determining for both reactions. Comparison of the sulfur elemental effect on V-max/K-m revealed differences in the mechanism by which either nucleotide is incorporated. Consistent with these data, an induced fit mechanism for nucleotide specificity is proposed whereby PAP couples a uniform binding mechanism, which selects for ATP, with a ground-state destabilization mechanism, which serves to accelerate the velocity for the correct substrate.