Teschner W, Serre M C, Garel J R
Laboratoire d'Enzymologie Centre, National de la Recherche Scientifique, Gif-Sur-Yvette, France.
Biochimie. 1990 Jan;72(1):33-40. doi: 10.1016/0300-9084(90)90170-l.
Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli. D-mannitol-1-phosphate dehydrogenase was purified to homogeneity from Escherichia coli, and its physicochemical and enzymatic properties were investigated. The molecular weight of the polypeptide chain is 45,000 as determined by polyacrylamide gel electrophoresis in denaturing conditions. High performance size exclusion chromatography gives an apparent molecular weight of 47,000 for the native enzyme, showing that D-mannitol-1-phosphate dehydrogenase is a monomeric NAD-dependent dehydrogenase. D-mannitol-1-phosphate dehydrogenase is rapidly denatured by 6 M guanidine hydrochloride. Non-superimposable transition curves for the loss of activity and the changes in fluorescence suggest the existence of a partially folded inactive intermediate. The protein can be fully renatured after complete unfolding, and the regain of both native fluorescence and activity occurs rapidly within a few seconds at pH 7.5 and 20 degrees C. Such a high rate of reactivation is unusual for a protein of this size. D-mannitol-1-phosphate dehydrogenase is specific for mannitol-1-phosphate (or fructose-6-phosphate) as a substrate and NAD+ (or NADH) as a cofactor. Zinc is not required for the activity. The affinity of D-mannitol-1-phosphate dehydrogenase for the reduced or oxidized form of its substrate or cofactor remains constant with pH. The affinity for NADH is 20-fold higher than for NAD+. The forward and reverse catalytic rate constants of the reaction: mannitol-1-phosphate + NAD+ in equilibrium fructose-6-phosphate + NADH have different pH dependences. The oxidation of mannitol-1-phosphate has an optimum pH of 9.5, while the reduction of fructose-6-phosphate has its maximum rate at pH 7.0. At pH values around neutrality the maximum rate of reduction of fructose-6-phosphate is much higher than that of oxidation of mannitol-1-phosphate. The enzymatic properties of isolated D-mannitol-1-phosphate dehydrogenase are discussed in relation to the role of this enzyme in the intracellular metabolism.
大肠杆菌中磷酸甘露醇-1-脱氢酶的酶学性质、复性及代谢作用。从大肠杆菌中纯化得到了具有同质性的D-磷酸甘露醇-1-脱氢酶，并对其理化性质和酶学性质进行了研究。在变性条件下通过聚丙烯酰胺凝胶电泳测定，该多肽链的分子量为45,000。高效尺寸排阻色谱法测得天然酶的表观分子量为47,000，表明D-磷酸甘露醇-1-脱氢酶是一种单体NAD依赖型脱氢酶。D-磷酸甘露醇-1-脱氢酶能被6M盐酸胍快速变性。活性丧失和荧光变化的非重叠过渡曲线表明存在部分折叠的无活性中间体。完全展开后，该蛋白质可完全复性，在pH 7.5和20℃条件下，几秒钟内即可迅速恢复天然荧光和活性。对于这种大小的蛋白质来说，如此高的再活化速率并不常见。D-磷酸甘露醇-1-脱氢酶对磷酸甘露醇-1-（或磷酸果糖-6-）作为底物以及NAD+（或NADH）作为辅因子具有特异性。该酶的活性不需要锌。D-磷酸甘露醇-1-脱氢酶对其底物或辅因子的还原型或氧化型的亲和力在不同pH值下保持恒定。对NADH的亲和力比对NAD+高20倍。反应：磷酸甘露醇-1- + NAD+ ⇌ 磷酸果糖-6- + NADH的正向和反向催化速率常数具有不同的pH依赖性。磷酸甘露醇-1-的氧化反应最适pH为9.5，而磷酸果糖-6-的还原反应在pH 7.0时速率最大。在接近中性的pH值下，磷酸果糖-6-的最大还原速率远高于磷酸甘露醇-1-的氧化速率。本文结合该酶在细胞内代谢中的作用，讨论了分离得到的D-磷酸甘露醇-1-脱氢酶的酶学性质。
