Slatner M, Nidetzky B, Kulbe K D
Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur Wien (BOKU), Austria.
Biochemistry. 1999 Aug 10;38(32):10489-98. doi: 10.1021/bi990327g.
To characterize catalysis by NAD-dependent long-chain mannitol 2-dehydrogenases (MDHs), the recombinant wild-type MDH from Pseudomonas fluorescens was overexpressed in Escherichia coli and purified. The enzyme is a functional monomer of 54 kDa, which does not contain Zn(2+) and has B-type stereospecificity with respect to hydride transfer from NADH. Analysis of initial velocity patterns together with product and substrate inhibition patterns and comparison of primary deuterium isotope effects on the apparent kinetic parameters, (D)k(cat), (D)(k(cat)/K(NADH)), and (D)(k(cat)/K(fructose)), show that MDH has an ordered kinetic mechanism at pH 8.2 in which NADH adds before D-fructose, and D-mannitol and NAD are released in that order. Isomerization of E-NAD to a form which interacts with D-mannitol nonproductively or dissociation of NAD from the binary complex after isomerization is the slowest step (>/=110 s(-)(1)) in D-fructose reduction at pH 8.2. Release of NADH from E-NADH (32 s(-)(1)) is the major rate-limiting step in mannitol oxidation at this pH. At the pH optimum for D-fructose reduction (pH 7.0), the rate of hydride transfer contributes significantly to rate limitation of the catalytic cascade and the overall reaction. (D)(k(cat)/K(fructose)) decreases from 2.57 at pH 7.0 to a value of </=1 above pH 9.6, corresponding to the pK of 9.34 observed in the pH profile of k(cat)/K(fructose). Therefore, hydride transfer is not pH-dependent, and D-fructose is not sticky at pH 7.0. A comparison of the kinetic data of MDH and mammalian sorbitol dehydrogenase, presumably involved in detoxification metabolism, is used to point out a physiological function of MDH in the oxidation of D-mannitol with high specificity and fluxional efficiency under prevailing reaction conditions in vivo.
为了表征依赖NAD的长链甘露醇2-脱氢酶(MDHs)的催化作用,来自荧光假单胞菌的重组野生型MDH在大肠杆菌中过表达并纯化。该酶是一种54 kDa的功能性单体,不含Zn(2+),在从NADH转移氢化物方面具有B型立体特异性。对初速度模式以及产物和底物抑制模式的分析,以及对初级氘同位素效应在表观动力学参数(D)k(cat)、(D)(k(cat)/K(NADH))和(D)(k(cat)/K(果糖))上的比较表明,MDH在pH 8.2时具有有序的动力学机制,其中NADH在D-果糖之前添加,D-甘露醇和NAD按该顺序释放。E-NAD异构化为与D-甘露醇非生产性相互作用的形式或异构化后NAD从二元复合物中解离是pH 8.2时D-果糖还原中最慢的步骤(≥110 s(-1))。在该pH下,E-NADH释放NADH(32 s(-1))是甘露醇氧化中的主要限速步骤。在D-果糖还原的最佳pH(pH 7.0)下,氢化物转移速率对催化级联和整体反应的速率限制有显著贡献。(D)(k(cat)/K(果糖))从pH 7.0时的2.57降至pH 9.6以上时≤1的值,对应于在k(cat)/K(果糖)的pH曲线中观察到的9.34的pK值。因此,氢化物转移不依赖于pH,并且D-果糖在pH 7.0时不具有粘性。对MDH和可能参与解毒代谢的哺乳动物山梨醇脱氢酶的动力学数据进行比较,以指出MDH在体内主要反应条件下以高特异性和通量效率氧化D-甘露醇的生理功能。