A study of the sulfhydryl groups of rat brain hexokinase.
A study of the sulfhydryl groups of rat brain hexokinase.
复制标题
大鼠脑己糖激酶的巯基的研究。
DOI：
10.1016/0003-9861(74)90469-x
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发表时间：
1974
影响因子：
3.9
通讯作者：
J. Wilson
中科院分区：
文献类型：
--
作者：
A. Chou;J. Wilson
大鼠脑已糖激酶(ATP：D-己糖-6-磷酸转移酶，EC 2.7.1.1)与5，5‘-二硫双-(2-硝基苯甲酸酯)反应迅速失活。在没有配体(25℃时k=0.641分钟−1)或饱和浓度的三磷酸腺苷(游离或与镁离子络合)或P1存在的情况下，失活遵循单相一级动力学，存在时失活率略有增加(k⋍0.7min−1)。相反，葡萄糖和葡萄糖-6-P显著降低了失活率；在这些配体存在下的失活是两相的，两种一级速率(k⋍0.5min−1和0.01min−1)是可区分的。该酶含有14个巯基，与5，5‘-二硫代-(2-硝基)苯甲酸酯反应；这些基团在天然酶中在25℃下反应2小时后完成，或与尿素或胍变性酶反应约5分钟后完成。在天然酶中，根据其与5，5‘-二硫代二(2-硝基)苯甲酸酯的快速(k⪢0.7min−1)、中间(k⋍0.5-0.7min−1)或慢(k⋍0.02min−1)反应速度，可将其区分为F-、I-或S类型。失活率与I型巯基反应速率的相关性表明，I型巯基包括催化活性所必需的残基。F型残基显然不是活性所必需的。测定了ATP、P_1、葡萄糖和葡萄糖-6-P对羟基反应活性的影响。在没有配体的情况下，可以区分S-、I-和F型的巯基。在这些配体的饱和浓度存在下，F、I和S类分别含有：含有三磷酸腺苷、1、4和7残基；含有P1、1、3和7残基；含有葡萄糖、1、2和5残基；含有葡萄糖-6-P、1、2和1残基。与这些配体存在下的失活速率常数的比较再次表明，I型巯基在维持酶活性方面特别重要。目前的结果表明，大鼠脑己糖激酶中的巯基残基的反应性与牛脑酶中的巯基很相似[VD Redkar和UW Kenkare(1972)，J.Biol]。化学，247,7576-7584]。
Rat brain hexokinase (ATP: d-hexose-6-phosphotransferase, EC 2.7. 1.1) is rapidly inactivated by reaction with 5, 5′-dithiobis-(2-nitrobenzoate). The inactivation follows monophasic first-order kinetics in either the absence of ligands (k= 0.641 min− 1 at 25° C) or in the presence of saturating levels of ATP (free or complexed with Mg 2+) or P 1; the inactivation rate is slightly increased (k⋍ 0.7 min− 1) in the presence of ATP or P 1. In contrast, glucose and glucose-6-P markedly decrease the inactivation rate; inactivation in the presence of these ligands is biphasic, with two first-order rates (k⋍ 0.5 min− 1 and 0.01 min− 1) being distinguishable. The enzyme contains 14 sulfhydryl groups which react with 5, 5′-dithiobis-(2-nitrobenzoate); reaction of these groups in the native enzyme is complete after 2 hr at 25° C, or in approx 5 min with the urea or guanidine-denatured enzyme. In the native enzyme, three classes of sulfhydryl groups are distinguishable and are designated as F-, I-, or S-type based on their fast (k⪢ 0.7 min− 1), intermediate (k⋍ 0.5-0.7 min− 1), or slow (k⋍ 0.02 min− 1 rates of reaction with 5, 5′-dithiobis-(2-nitrobenzoate). The correlation of inactivation rates with the rates for reaction of the I-type sulfhydryls indicates that the I-type sulfhydryls include residues necessary for catalytic activity. The F-type residues are clearly not required for activity. The effects of ATP, P 1, glucose, and glucose-6-P on the reactivity of the sulfhydryls have been determined. As in the absence of ligands, S-, I-, and F-type sulfhydryls could be distinguished. In the presence of saturating concentrations of these ligands, the F, I, and S classes of sulfhydryls contained respectively: with ATP, 1, 4, and 7 residues; with P 1, 1, 3, and 7 residues; with glucose, 1, 2, and 5 residues; with glucose-6-P, 1, 2, and 1 residues. Comparison with rate constants for inactivation in the presence of these ligands again indicated that I-type sulfhydryls were particularly important in maintenance of enzyme activity. The present results indicate considerable similarity between the reactivity of the sulfhydryl residues in rat brain hexokinase and the sulfhydryls of the bovine brain enzyme [VD Redkar and UW Kenkare (1972), J. Biol. Chem., 247, 7576–7584].