Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase:: relevance of arginine 70 for catalysis
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase:: relevance of arginine 70 for catalysis
复制标题
DOI：
10.1016/j.biochi.2004.06.001
复制
发表时间：
2004-06-01
期刊：
影响因子：
3.9
通讯作者：
Cardemil, E
中科院分区：
文献类型：
--
作者：
Ravanal, MC;Flores, M;Cardemil, E
酿酒酵母磷酸烯醇丙酮酸（PEP）羧激酶是酿酒酵母代谢途径中的关键酶，在二价金属离子存在下催化草酰乙酸脱羧和ATP的γ-磷酰基转移，生成PEP、ADP和CO2。已有的实验表明，金属位点I氨基酸残基的突变降低了酶的催化效率和蛋白对PEP的亲和力。证明PEP和金属离子第一配位层的水分子之间的氢键相互作用对于催化的相关性[Biochemistry 41（2002）12763]。为了进一步了解位于PEP结合位点的氨基酸残基的功能，我们现在讨论了Arg（70）的催化重要性，其胍基靠近PEP羧基。Arg（70）Lys PEP羧激酶的动力学参数几乎不变，而Arg 70 Gln和Arg 70 Met突变酶的催化效率降低了4-5个数量级。为了评估酶与PEP的相互作用，将野生型的磷酸吡啶氧基衍生物Arg 70 Lys、Arg 70 Gln和Arg 70 Met S.制备了啤酒酵母PEP羧激酶探针，并利用探针与PEP结合后荧光发射的变化，获得了相应衍生酶-PEP-Mn ~（2+）复合物的解离平衡常数。滴定实验表明，在Arg 70 Met和Arg 70 Gln突变酶中产生PEP结合亲和力的2.1 kcal/mol损失。有人提出，精氨酸（70）的胍基团和PEP的羧基之间的静电相互作用是重要的PEP绑定和进一步的步骤中的催化。(C)2004年，Elsevier SAS。All rights reserved.
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase is a key enzyme of the gluconeogenic pathway and catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2 in the presence of a divalent metal ion. Previous experiments indicate that mutation of amino acid residues at metal site I decrease the enzyme catalytic efficiency and the affinity of the protein for PEP. evidencing the relevance of hydrogen-bond interactions between PEP and water molecules of the first coordination sphere of the metal ion for catalysis [Biochemistry 41 (2002) 12763]. To further understand the function of amino acid residues located in the PEP binding site, we have now addressed the catalytic importance of Arg(70), whose guanidinium group is close to the PEP carboxyl group. Arg(70)) mutants of PEP carboxykinase were prepared, and almost unaltered kinetic parameters were found for the Arg(70)Lys PEP carboxykinase, while a decrease in 4-5 orders of magnitude for the catalytic efficiency was detected for the Arg70Gln and Arg70Met altered enzymes. To evaluate the enzyme interaction with PEP, the phosphopyridoxyl-derivatives of wild type, Arg70Lys, Arg70Gln, and Arg70Met S. cerevisiae PEP carboxykinase were prepared, and the change in the fluorescence emission of the probe upon PEP binding was used to obtain the dissociation equilibrium constant of the corresponding derivatized enzyme-PEP-Mn2+ comp lex. The titration experiments showed that a loss in 2.1 kcal/mol in PEP binding affinity is produced in the Arg70Met and Arg70Gln mutant enzymes. It is proposed that the electrostatic interaction between the guanidinium group of Arg(70) and the carboxyl group of PEP is important for PEP binding and for further steps in catalysis. (C) 2004 Elsevier SAS. All rights reserved.