STRUCTURAL STUDY OF ENZYMIC PO3 TRANSFER--ACETATE KINASE

磷酸化基团的酶转移是控制许多细胞过程的核心。诸如蛋白激酶、G蛋白、小gtp酶和atp酶等酶的催化循环对于这些分子调节自身和其他蛋白质的能力至关重要。单个酶的催化活性失常可直接导致疾病。然而，对这些酶的调控机制还缺乏详细的了解，因为尽管进行了大量的研究，磷酸化转移的机制仍然不完全清楚。醋酸激酶是一种具有独特催化性能的酶。对其结构和功能的研究可能为酶催化的磷酰基转移化学提供新的认识。在谷氨酰残基上磷酸化的酶的一种形式具有催化中共价中间体的基本特征。然而，磷基是通过构型反转转移的；这种立体化学的变化通常被认为是磷酸盐在没有酶联共价中间体的情况下从底物直接在线转移到产物的证据（通常是唯一的证据）。这两种证据可以调和吗？这个问题的答案对于理解酶促磷酸化转移至关重要。该项目的长期目标是阐明醋酸激酶的机制，基于这些结果重新思考其他酶的磷酸化转移机制，了解该家族酶结构和功能的进化，并最终评估醋酸激酶机制对细菌生理的影响。在提议的项目期间，来自Methanosarcina thermoophila的热稳定醋酸激酶的结构将在各种底物和抑制剂的存在下通过x射线晶体学确定。相对稳定的共价催化中间体的结构将被确定。与其他物种的乙酸激酶和丁酸激酶的结构比较研究将有助于解释活性位点结构，并将提供有关该酶家族底物特异性和嗜热性的结构基础的信息。对催化至关重要的残基将通过检查晶体结构和诱变实验来确定；有趣的突变体将进行生化和结构分析。从醋酸激酶的研究中获得的原理将扩展到其他磷酸转移酶，从己糖激酶开始。所提出的研究应提供醋酸激酶的机制的阐明，这将对许多其他酶的磷酸化转移的理解具有指导意义。

The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. The catalytic cycles of enzymes such as protein kinases, G proteins, small GTPases, and ATPases are crucial to the ability of these molecules to regulate themselves and other proteins. Aberrations in catalytic activity of a single enzyme can result directly in disease. Detailed understanding of the regulatory mechanisms employed by such enzymes is lacking, however, because despite intense study, the mechanism of phosphoryl transfer is still incompletely understood. Acetate kinase is an enzyme with unique catalytic properties. The study of its structure and function may provide new insights into the chemistry of enzyme-catalyzed phosphoryl group transfer. A form of the enzyme phosphorylated on a glutamyl residue has essential hallmarks of a covalent intermediate in catalysis. Yet the phosphoryl group is transferred with inversion of configuration; this change in stereochemistry is typically taken as evidence (often the sole evidence) for a direct, in-line transfer of phosphate from substrate to product without an enzyme-linked covalent intermediate. Can these two lines of evidence be reconciled? The answer to this question is critical to a general understanding enzymatic phosphoryl transfer. The long-term goal of this project is to elucidate the mechanism of acetate kinase, to rethink the mechanisms of phosphoryl transfer in other enzymes based on these results, to understand the evolution of enzyme structure and function in this family, and, eventually, to assess the ramifications of the acetate- kinase mechanism for bacterial physiology. In the proposed project period, the structure of the thermostable acetate kinase from Methanosarcina thermophila will be determined by X-ray crystallography in the presence of various combinations of substrates and inhibitors. The structure of the relatively stable covalent catalytic intermediate will be determined. Comparative structural studies with acetate kinase and butyrate kinase from other species will aid in the interpretation of active-site structures, and will provide information about the structural basis of substrate specificity and thermophily in this enzyme family. Residues critical to catalysis will be identified by inspection of the crystal structures and mutagenesis experiments; interesting mutants will be analyzed biochemically and structurally. Principles learned from the study of acetate kinase will be extended to other phosphotransferase, beginning with hexokinase. The proposed studies should provide an elucidation of the mechanism of acetate kinase, which will have implications for the understanding of phosphoryl transfer in many other enzymes.