Lindstad R I, McKinley-McKee J S
Institute of Biochemistry, University of Oslo, Norway.
Eur J Biochem. 1996 Oct 1;241(1):142-8. doi: 10.1111/j.1432-1033.1996.0142t.x.
Reversible inhibition of sheep liver sorbitol dehydrogenase by various thiol compounds has been studied. Most species inhibit the enzyme-catalyzed reaction competitively with respect to sorbitol, due to the formation of ternary enzyme-NAD-thiol complexes. The primary interaction of thiol inhibitors with the enzyme active site involves the catalytic zinc atom, and a bidentate mode of binding to the active site metal is indicated for some bifunctional thiols in their ternary complexes. Enzyme-bound thiolate facilitates NAD binding to the enzyme and vice versa, mainly due to mutual electrostatic stabilization. The aromatic thiols 1-thio-1-phenylmethane and 1-thio-2-phenylethane are especially potent inhibitors with an inhibition constant of 0.30 microM at pH 9.9. The inhibitory effect of aliphatic thiols, which is positively correlated with alkyl chain length, parallels that observed previously with the related enzyme horse liver alcohol dehydrogenase and indicates that interaction with an enzymic hydrophobic site is important for inhibitor binding. Several reversible inhibitors afford competitive protection against affinity labelling of the enzyme by 2-bromo-3-(5-imidazolyl) propionic acid due to the formation of binary enzyme-thiol complexes. The present study establishes thionucleosides as a novel class of potent sorbitol dehydrogenase inhibitors. The thionucleosides 6-thioguanosine and 6-thioinosine gave mixed inhibition with respect to sorbitol, due to the formation of enzyme-NAD-inhibitor and enzyme-NADH-inhibitor complexes. In order to enable a correlation of the substrate and inhibitor specificities of the enzyme, the kinetic constants for several sorbitol dehydrogenase substrates were determined. L-threitol and DL-1-phenyl-1,2-ethanediol are good substrates with, at high pH, kinetic constants similar to those of sorbitol. The potent inhibition by dithiothreitol and the aromatic thiols thus parallels the substrate specificity of the enzyme. The sorbitol competitive inhibitor 1-thiosorbitol is also a substrate with, at pH 7.4, a maximum velocity of 0.17 s-1 and a Michaelis constant of 8.6 mM. Dithiothreitol forms a tight ternary complex with the enzyme-NAD complex with a molar absorbance of 16.4 x 10(3) M-1 . cm-1 at 311 nm. A spectrophotometric titration of the enzyme with NAD in the presence of dithiothreitol is described, which enables an accurate determination of the concentration of sorbitol dehydrogenase active sites and confirms the activity assay of the enzyme.
已对各种硫醇化合物对绵羊肝脏山梨醇脱氢酶的可逆抑制作用进行了研究。大多数此类化合物对该酶催化的反应具有竞争性抑制作用，且作用对象是山梨醇，这是由于形成了三元酶 - 烟酰胺腺嘌呤二核苷酸（NAD） - 硫醇复合物。硫醇抑制剂与酶活性位点的主要相互作用涉及催化性锌原子，并且对于某些双功能硫醇在其三元复合物中与活性位点金属的结合方式表明为双齿模式。与酶结合的硫醇盐促进NAD与酶的结合，反之亦然，这主要是由于相互的静电稳定作用。芳族硫醇1 - 硫代 - 1 - 苯基甲烷和1 - 硫代 - 2 - 苯基乙烷是特别有效的抑制剂，在pH 9.9时抑制常数为0.30微摩尔。脂肪族硫醇的抑制作用与烷基链长度呈正相关，这与之前在相关酶马肝醇脱氢酶中观察到的情况相似，表明与酶的疏水位点相互作用对于抑制剂结合很重要。几种可逆抑制剂由于形成了二元酶 - 硫醇复合物，从而对2 - 溴 - 3 -（5 - 咪唑基）丙酸对该酶的亲和标记提供了竞争性保护。本研究确定硫代核苷是一类新型的有效山梨醇脱氢酶抑制剂。硫代核苷6 - 硫鸟苷和6 - 硫肌苷对山梨醇产生混合抑制作用，这是由于形成了酶 - NAD - 抑制剂和酶 - NADH - 抑制剂复合物。为了能够关联该酶的底物和抑制剂特异性，测定了几种山梨醇脱氢酶底物的动力学常数。L - 苏糖醇和DL - 1 - 苯基 - 1,2 - 乙二醇是良好的底物，在高pH值下，其动力学常数与山梨醇的相似。二硫苏糖醇和芳族硫醇的强效抑制作用因此与该酶的底物特异性相似。山梨醇竞争性抑制剂1 - 硫代山梨醇也是一种底物，在pH 7.4时，最大反应速度为0.17 s⁻¹，米氏常数为8.6 mM。二硫苏糖醇与酶 - NAD复合物形成紧密的三元复合物，在311 nm处摩尔吸光度为16.4×10³ M⁻¹·cm⁻¹。描述了在二硫苏糖醇存在下用NAD对该酶进行分光光度滴定，这能够准确测定山梨醇脱氢酶活性位点的浓度并确认该酶的活性测定。
