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中心。蛋白激酶、G蛋白、小GTP酶和ATPase等酶的催化循环对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，对这类酶的调节机制缺乏详细的了解，因为尽管进行了大量的研究，但对磷酸基转移的机制仍然不完全了解。醋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可能为酶催化的磷酸基转移提供新的认识。在谷氨酰残基上磷酸化的一种酶具有催化中共价中间体的基本特征。然而，磷酰基是随着构型的倒置而转移的；这种立体化学的变化通常被认为是磷酸盐从底物到产物的直接、在线转移的证据(通常是唯一的证据)，而不需要酶连接的共价中间体。这两种证据可以调和吗？这个问题的答案对于全面理解酶促磷酸转移是至关重要的。该项目的长期目标是阐明醋酸激酶的机制，根据这些结果重新思考其他酶中的磷酸转移机制，了解该家族中酶结构和功能的演变，并最终评估醋酸激酶机制对细菌生理学的影响。在拟议的项目期内，将在各种底物和抑制剂组合存在的情况下，通过X射线结晶学确定嗜热甲烷八叠球菌耐热醋酸激酶的结构。将确定相对稳定的共价催化中间体的结构。与其他物种的醋酸激酶和丁酸激酶的比较结构研究将有助于解释活性部位结构，并将提供关于该酶家族底物专一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学到的原理将扩展到其他磷酸转移酶，从己糖激酶开始。这项研究将有助于阐明醋酸激酶的作用机制，这将对理解许多其他酶中的磷酸基转移具有重要意义。

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.