Kinetic and structural effects of activation of bovine kidney aldose reductase.
Kinetic and structural effects of activation of bovine kidney aldose reductase.
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牛肾醛糖还原酶激活的动力学和结构效应。
DOI：
10.1021/bi00439a006
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Mathur,EJ
中科院分区：
文献类型：
--
作者：
Grimshaw,CE;Shahbaz,M;Jahangiri,G;Putney,CG;McKercher,SR;Mathur,EJ
从牛肾中提纯的醛糖还原酶，在依赖温度的过程中从低Ģm/低形式转变为高A^/高K^形式。用荧光光谱、圆二色谱和5，5‘-二硫代二(2-硝基苯甲酸)修饰硫醇检测到的激活对酶的表观mt、p或均一性没有影响，用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和琼脂糖等电聚焦检测。对于一系列具有酶激活状态的乙醛底物，V^的变化不到3倍，在酶激活时平均增加了(17ą4)倍。根据底物的不同，V/K Mehyie的增减幅度最高可达4倍。激活使酶对醛糖还原酶抑制剂的抑制不敏感，表观K值由依帕瑞司[ONO-2 2 35，(Ģ)-3-(carboxymethyl)-(Ģ)-5-[2-methyl-3-phenylpropenylidene]-rhodanine]]的2倍增加到AL-15 76(螺环[2，7-二氟-9，4/咪唑烷]-2，5‘-二酮)的2 0 0倍。在激活过程中观察到的醛底物的双相双互易图和AL-1576和Statil[ICI-128436；3-[(4-bromo-2-fluorobenzyl)-4-oxo-377-phthalazin-l-ylacetic酸]抑制的双相Dixon图，都是由这两种酶的各自贡献来定量解释的。在动力学数据分析的基础上，提出了游离酶的异构化限制了未活化的酶的前向反应速度的机理，它是受激活影响的主要步骤。
Aldose reductase, purified to homogeneity from bovine kidney, is converted in a temperature-dependent process from a low-Ģm/low- form to a high-A^/high-K^ form of the enzyme. Activation, which results in significant changes in the protein secondary structure, as detected by fluorescence spec-troscopy, circular dichroism, and thiol modification with 5, 5'-dithiobis (2-nitrobenzoic acid), has no effect on the apparent Mt, p/, or homogeneity of the enzyme, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose isoelectric focusing. V^, which varied less than 3-fold for a series of aldehyde substrates with either activation state of the enzyme, increased an average of (17 ą 4)-fold upon activation of the enzyme. V/KMehyie increased or decreased up to 4-fold, depending on the substrate. Activation desensitized the enzyme to inhibition by aldose reductase inhibitors, with the apparent K\value increasing from 2-fold for Epalrestat [ONO-2235,(Ģ)-3-(carboxymethyl)-(Ģ)-5-[2-methyl-3-phenylpropenylidene]-rhodanine] to 200-fold for AL-1576 (spiro [2, 7-difluorofluorene-9, 4/-imidazolidine]-2,, 5'-dione). Biphasic double-reciprocal plots for the aldehyde substrates and biphasic Dixon plots for inhibition by AL-1576 and Statil [ICI-128 436; 3-[(4-bromo-2-fluorobenzyl)-4-oxo-377-phthalazin-l-ylacetic acid], observed during the course of activation, are quantitatively accounted for bythe individual contributions of the two enzyme forms. On the basis of an analysis of the kinetic data, a mechanism is proposed in which isomerization of the free enzyme limits the rate of the forward reactionfor the unactivated enzyme and is the primary step affected by activation.