Young M C, Kuhl S B, von Hippel P H
Institute of Molecular Biology, University of Oregon, Eugene 97402.
J Mol Biol. 1994 Feb 4;235(5):1436-46. doi: 10.1006/jmbi.1994.1099.
A theory is presented to describe the coupling of the directional movement of ATPase-containing translocases (such as helicases) along polymeric lattices to the steady-state kinetic parameters of the ATPase activity that drives this movement. This theory was developed to explain the results of an experimental investigation of one such enzyme, the bacteriophage T4 gene 41 protein helicase. The salient feature of the theory is that it correctly predicts the dependence of the rate of ATP hydrolysis by ATP-driven translocases on the length of polymer lattices along which they move. In the steady-state rate equation: [formula: see text] either Vmax, or K(act), or both, may depend on the lattice length. Two translocation models are considered. The first is a simple mechanism of the type E<-->E-Lat-->E, where the E-Lat-->E step represents the sum of the translocation steps of the enzyme along, and enzyme release from, the lattice. In the second model this mechanism is expanded to add an additional kinetic step, either before or after the translocation process. Variants of this second model can be used to represent the most simple translocase mechanisms. Another method of measuring the lattice length dependence of an ATP-driven translocase, which is applicable particularly to ATPases moving along DNA lattices, involves the use of lattice-binding proteins (such as single-stranded DNA binding proteins) that can block the movement of the translocases and therefore simulate lattice ends. In this protocol the dependence of the ATPase kinetics of the translocase on lattice length can be studied by experiments on long lattices complexed with lattice-binding proteins to various binding densities. This method is not always as unambiguous as direct measurement of ATPase activity on lattices of defined length, but can help to discriminate between mechanisms. The significance of the steady-state kinetic parameters obtained in such experiments is discussed in terms of the mechanistic rate constants that define the models we have investigated.
本文提出了一种理论，用于描述含ATP酶的转位酶（如解旋酶）沿聚合物晶格的定向运动与驱动该运动的ATP酶活性的稳态动力学参数之间的耦合关系。该理论是为了解释对一种这样的酶——噬菌体T4基因41蛋白解旋酶的实验研究结果而发展起来的。该理论的显著特点是它能正确预测ATP驱动的转位酶的ATP水解速率对其移动所沿聚合物晶格长度的依赖性。在稳态速率方程：[公式：见原文]中，要么Vmax，要么K(act)，或者两者都可能取决于晶格长度。考虑了两种转位模型。第一种是E<-->E-Lat-->E类型的简单机制，其中E-Lat-->E步骤代表酶沿晶格的转位步骤以及从晶格释放的总和。在第二种模型中，该机制被扩展以在转位过程之前或之后添加一个额外的动力学步骤。这种第二种模型的变体可用于表示最简单的转位酶机制。另一种测量ATP驱动的转位酶对晶格长度依赖性的方法，特别适用于沿DNA晶格移动的ATP酶，涉及使用晶格结合蛋白（如单链DNA结合蛋白），其可阻断转位酶的移动，从而模拟晶格末端。在此方案中，通过对与晶格结合蛋白以各种结合密度复合的长晶格进行实验，可以研究转位酶的ATP酶动力学对晶格长度的依赖性。这种方法并不总是像在确定长度的晶格上直接测量ATP酶活性那样明确，但有助于区分不同机制。根据定义我们所研究模型的机制速率常数，讨论了在此类实验中获得的稳态动力学参数的意义。
