Nar H, Huber R, Auerbach G, Fischer M, Hösl C, Ritz H, Bracher A, Meining W, Eberhardt S, Bacher A
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12120-5. doi: 10.1073/pnas.92.26.12120.
GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside. The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135.
大肠杆菌的GTP环化水解酶I是一种具有D5对称性的环状同十聚体，催化一个复杂的环扩展反应，该反应有利于从GTP形成二氢新蝶呤三磷酸。在3埃的分辨率下确定了该酶与结合在活性位点的底物类似物dGTP的复合物的X射线结构。在十聚体中，存在10个等效的活性位点，每个活性位点都包含一个由3个相邻亚基的表面区域形成的10埃深的口袋。底物与蛋白质形成复杂的氢键网络。通过定点诱变修饰活性位点残基，并测量突变蛋白的酶活性。在此基础上，提出了酶催化反应的机制。咪唑环的裂解由His-179使N7质子化引发，随后水进攻嘌呤系统的C8。半胱氨酸Cys-110 Cys-181可能参与这一反应步骤。咪唑环的打开可能与呋喃糖环的裂解协同进行，从糖苷生成席夫碱。GTP的γ-磷酸可能通过激活Ser-135的羟基参与随后碳水化合物侧链的阿马多里重排。
