Hara A, Shinoda M, Kanazu T, Nakayama T, Deyashiki Y, Sawada H
Department of Biochemistry, Gifu Pharmaceutical University, Japan.
Biochem J. 1991 Apr 1;275 ( Pt 1)(Pt 1):121-6. doi: 10.1042/bj2750121.
The dehydrogenase activity of dimeric dihydrodiol dehydrogenases (DD) purified from pig and rabbit lenses was inhibited by either L-ascorbic acid or its epimer, isoascorbic acid, at pH 7.5. Isoascorbate [IC50 (concn. giving 50% inhibition) = 0.043 mM for the pig enzyme; IC50 = 0.13 mM for the rabbit enzyme] was a more potent inhibitor than ascorbate (IC50 values 0.45 and 0.90 mM respectively), but 1 mM-dehydroascorbate gave less than 30% inhibition. Glucose, glucuronate, gulono-gamma-lactone, glutathione and dithiothreitol did not inhibit the enzyme activity. The inhibition by isoascorbate and ascorbate was instantaneous and reversible, and their inhibitory potency was decreased by addition of ascorbate oxidase. In the reverse reaction, isoascorbate and ascorbate gave low IC50 values of 0.013 and 0.10 mM respectively for the pig enzyme and 0.025 and 0.25 mM for the rabbit enzyme. The inhibition patterns by the two compounds were competitive with respect to dihydrodiols of naphthalene and benzene and uncompetitive with respect to NADP+, but those in the reverse reaction were uncompetitive with respect to both carbonyl substrate and NADPH. The steady-state kinetic measurements in the forward and reverse reactions by the pig enzyme were consistent with an ordered Bi Bi mechanism, in which NADP+ binds to the enzyme first and NADPH leaves last. The results indicate that ascorbate and its epimer directly bind to an enzyme: NADP+ binary complex as dead-end inhibitors. Thus ascorbate may be an important modulator of DD in the lens.
从猪和兔晶状体中纯化得到的二聚体二氢二醇脱氢酶（DD）的脱氢酶活性，在pH 7.5时受到L-抗坏血酸或其差向异构体异抗坏血酸的抑制。异抗坏血酸盐[猪酶的IC50（产生50%抑制的浓度）= 0.043 mM；兔酶的IC50 = 0.13 mM]是比抗坏血酸盐（IC50值分别为0.45和0.90 mM）更强效的抑制剂，但1 mM脱氢抗坏血酸的抑制率不到30%。葡萄糖、葡糖醛酸、古洛糖酸-γ-内酯、谷胱甘肽和二硫苏糖醇不抑制酶活性。异抗坏血酸盐和抗坏血酸盐的抑制是瞬时且可逆的，添加抗坏血酸氧化酶会降低它们的抑制效力。在逆反应中，异抗坏血酸盐和抗坏血酸盐对猪酶的IC50值分别低至0.013和0.10 mM，对兔酶分别为0.025和0.25 mM。这两种化合物的抑制模式对于萘和苯的二氢二醇是竞争性的，对于NADP +是非竞争性的，但在逆反应中对于羰基底物和NADPH都是非竞争性的。猪酶在正向和逆反应中的稳态动力学测量结果与有序的Bi Bi机制一致，其中NADP +首先与酶结合，NADPH最后离开。结果表明抗坏血酸及其差向异构体作为无效抑制剂直接结合到酶：NADP +二元复合物上。因此，抗坏血酸可能是晶状体中DD的重要调节剂。
