STRUCTURAL STUDY OF ENZYMIC PO3 TRANSFER--ACETATE KINASE

磷酰基的酶促转移是许多细胞过程控制的核心。 酶如蛋白激酶、G蛋白、小GTP酶和ATP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。 单一酶催化活性的异常可直接导致疾病。 然而，缺乏对这些酶所采用的调节机制的详细了解，因为尽管进行了深入的研究，磷酰基转移的机制仍然不完全清楚。乙酸激酶是一种具有独特催化性质的酶。对其结构和功能的研究将为酶催化磷酰基转移反应的化学研究提供新的思路。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，磷酰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从底物直接在线转移到产物而无需酶联共价中间体的证据（通常是唯一的证据）。 这两条线索能协调一致吗？ 这个问题的答案是至关重要的一般理解酶磷酰基转移。该项目的长期目标是阐明乙酸激酶的机制，基于这些结果重新思考其他酶中磷酰基转移的机制，了解该家族中酶结构和功能的演变，并最终评估乙酸激酶机制对细菌生理学的影响。 在拟议的项目期间，将在存在各种底物和抑制剂组合的情况下，通过X射线晶体学确定来自嗜热甲烷八叠球菌的热稳定乙酸激酶的结构。 将确定相对稳定的共价催化中间体的结构。与其他物种的乙酸激酶和丁酸激酶的比较结构研究将有助于解释活性位点的结构，并将提供有关该酶家族的底物特异性和嗜热性的结构基础的信息。 将通过检查晶体结构和诱变实验来鉴定对催化作用至关重要的残基;将对感兴趣的突变体进行生化和结构分析。 从乙酸激酶研究中学到的原理将从己糖激酶开始扩展到其他磷酸转移酶。 拟议的研究应提供一个阐明的机制，乙酸激酶，这将有影响的理解磷酰基转移在许多其他酶。

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.