Marini I, Bucchioni L, Borella P, Del Corso A, Mura U
Dipartimento di Fisiologia e Biochimica, Università di Pisa, Italy.
Arch Biochem Biophys. 1997 Apr 15;340(2):383-91. doi: 10.1006/abbi.1997.9882.
Bovine lens sorbitol dehydrogenase (L-iditol:NAD+ 2-oxidoreductase, EC 1.1.1.14) (SDH) was purified to electrophoretic homogeneity (51 U/mg of protein) and characterized for both kinetic and some structural properties. The enzyme proves to be a homotetramer of 156 kDa containing one equivalent of zinc ion per subunit. Metal chelators such as EDTA and 1,10-phenanthroline determine a loss of enzyme activity which can be specifically recovered by addition of either zinc or manganese ions. Inactivation induced not only by metal chelators but also by thiol reagents is effectively prevented by the pyridine cofactor. Bovine lens SDH is active on polyalcohols and keto-sugars with more than three carbon atoms, and also requires special steric constraints for substrate recognition. Of the polyols, xylitol is the most effective substrate (kcat/KM of 8.1 s-1 mM-1), followed by sorbitol (kcat/KM of 1.59 s-1 mM-1); fructose, the most effective carbonyl substrate, displays a kcat/KM of only 0.9 s-1 mM-1. Analysis at the steady state of initial velocities as a function of the concentration of different substrates and cofactors and studies of product inhibition indicate for both fructose reduction and sorbitol oxidation a Theorell and Chance-type kinetic mechanism of action.
牛晶状体山梨醇脱氢酶（L-艾杜糖醇：NAD⁺ 2-氧化还原酶，EC 1.1.1.14）（SDH）被纯化至电泳纯（51 U/mg蛋白质），并对其动力学和一些结构特性进行了表征。该酶被证明是一种156 kDa的同四聚体，每个亚基含有一个锌离子。金属螯合剂如EDTA和1,10-菲咯啉会导致酶活性丧失，而添加锌离子或锰离子可特异性恢复酶活性。吡啶辅因子可有效防止不仅由金属螯合剂而且由硫醇试剂诱导的失活。牛晶状体SDH对含有三个以上碳原子的多元醇和酮糖具有活性，并且底物识别还需要特殊的空间限制。在多元醇中，木糖醇是最有效的底物（kcat/KM为8.1 s⁻¹ mM⁻¹），其次是山梨醇（kcat/KM为1.59 s⁻¹ mM⁻¹）；最有效的羰基底物果糖，其kcat/KM仅为0.9 s⁻¹ mM⁻¹。对不同底物和辅因子浓度的初始速度稳态分析以及产物抑制研究表明，果糖还原和山梨醇氧化均具有Theorell和Chance型动力学作用机制。
