Kinetic and chemical mechanisms of the sheep liver 6-phosphogluconate dehydrogenase
Kinetic and chemical mechanisms of the sheep liver 6-phosphogluconate dehydrogenase
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DOI：
10.1006/abbi.1996.0551
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发表时间：
1996-12-15
影响因子：
3.9
通讯作者：
Cook, PF
中科院分区：
文献类型：
--
作者：
Price, NE;Cook, PF
一个完整的动力学表征羊肝6-磷酸葡萄糖酸脱氢酶，包括产品和死端抑制模式，主要的氘同位素效应，和pH依赖性的动力学参数已经完成，以确定的动力学机制，并获得信息的化学机制的酶。提出了一种快速平衡随机动力学机制，产物抑制和死端抑制模式都是对称的，核酮糖B-磷酸和6-磺基葡萄糖酸都与6-磷酸葡萄糖酸（6-PG）竞争而与NADP非竞争，NADPH和ATP-核糖都与NADP竞争而与6-磷酸葡萄糖酸非竞争，1.5-2的等一级氘同位素效应对V-D，V-D/K-NADP，和V-D/K-6-PG与3-氘代-6-PG证实了快速平衡无规机理，并表明氢化物转移在整个反应中至少部分是速率限制的。最大速率依赖于pH，在低和高pH下分别以1和-1的斜率降低，pK值为6.4和8.6，V/K-NADP和V/K-6-PG也在低pH和高pH下以1和-1的斜率降低，分别给出pK值6.8和8.7以及6.9和7.8，pH速率曲线与催化残基参与结合的一般酸/一般碱机制一致，提出了一般酸和一般碱之间的反向质子化状态，其中未质子化的一般碱接受来自6-PG的C-3羟基的质子，伴随着氢化物转移，随后是所得3-PG的脱羧。酮中间体，得到烯醇化物，其被一般酸质子化，形成核酮糖5-磷酸。抑制性类似物5-磷酸核糖酸的pK（i）曲线的pH依赖性在低pH和高pH下分别以I和-1的斜率降低，并且pK为6.2和7.4，并且表明在V/K曲线中观察到固有pK。6-PG复合物似乎受到扰动，使得总碱从7.4-7.8降低到6.4-6.8的值，与6-PG直接作用使总酸的pK值由6.2-6.9提高到8.6-8.7。数据解释方面公布的晶体结构的E：6-PG，E：NADP，和E：NADPH复合物。(C)出版社：Academic Press，Inc.
A complete kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism and obtain information on the chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric, Ribulose B-phosphate and 6-sulfogluconate are both competitive with 6-phosphogluconate (6-PG) and noncompetitive with NADP, and NADPH and ATP-ribose are both competitive with NADP and noncompetitive with 6-phosphogluconate, Equal primary deuterium isotope effects of 1.5-2 on V-D, V-D/K-NADP, and V-D/K-6-PG with 3-deuterio-6-PG confirm a rapid equilibrium random mechanism and show that hydride transfer is at least partially rate limiting in the overall reaction, The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and -1, respectively, and pK values of 6.4 and 8.6, The V/K-NADP and V/K-6-PG also decrease at low and high pH with slopes of 1 and -1, giving pK values of 6.8 and 8.7 and of 6.9 and 7.8, respectively, The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding, Reverse protonation states between the general acid and the general base are proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6-PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to ave an enediol which is protonated by the general acid to form ribulose 5-phosphate. The pH dependence of the pK(i) profile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of I and -1, respectively, and pKs of 6.2 and 7.4 and suggests that intrinsic pKs are observed in the V/K profiles, The pKs of both the general base and general acid in the E:6-PG complex appears to be perturbed such that the general base decreases from 7.4-7.8 to a value of 6.4-6.8, and the pK of the general acid increases from 6.2-6.9 to a value of 8.6-8.7, as a result of direct interaction with 6PG. Data are interpreted with regard to the published crystal structures of the E:6-PG, E:NADP, and E:NADPH complexes. (C) 1996 Academic Press, Inc.