O'Brien M M, Schofield P J, Edwards M R
Biochem J. 1983 Apr 1;211(1):81-90. doi: 10.1042/bj2110081.
Sorbitol dehydrogenase was isolated from human brain and purified 690-fold, giving a final specific activity of 11.1 units/mg of protein. The enzyme preparation was nearly homogeneous, but was unstable at most temperatures. It exhibited a broad pH optimum of 7.5-9.0 in the forward reaction (i.e. sorbitol leads to fructose), and of 7.0 in the reverse reaction (i.e. fructose leads to sorbitol). Substrate-specificity studies demonstrated that the enzyme had the capability to oxidize a wide range of polyols and that the enzyme had a higher affinity for substrates in the forward reaction than in the reverse reaction, e.g. Km for sorbitol was 0.45 mM, and that for fructose was 480 mM. However, the Vmax. was 10 times greater in the reverse reaction. At high concentrations of fructose (500 mM) the enzyme exhibited substrate inhibition in the reverse reaction. The enzyme mechanism was sequential, as determined by the kinetic patterns arising from varying the substrate concentrations. In addition, both fructose and NADH protected the enzyme against thermal inactivation. These findings, together with product-inhibition data, suggested that the mechanism is random rapid equilibrium with two dead-end complexes.
山梨醇脱氢酶从人脑中分离出来，纯化了690倍，最终比活性为11.1单位/毫克蛋白质。该酶制剂几乎是纯的，但在大多数温度下不稳定。在正向反应（即山梨醇生成果糖）中，其最适pH范围较宽，为7.5 - 9.0，而在逆向反应（即果糖生成山梨醇）中，最适pH为7.0。底物特异性研究表明，该酶能够氧化多种多元醇，并且该酶对正向反应底物的亲和力高于逆向反应，例如山梨醇的Km值为0.45 mM，果糖的Km值为480 mM。然而，逆向反应的Vmax值大10倍。在高浓度果糖（500 mM）时，该酶在逆向反应中表现出底物抑制作用。根据改变底物浓度产生的动力学模式确定，该酶的作用机制是顺序性的。此外，果糖和NADH都能保护该酶免受热失活。这些发现，连同产物抑制数据，表明其机制是具有两个终止复合物的随机快速平衡。
