Department of Chemistry, Youngstown State University, Youngstown, Ohio 44555, USA.
J Biol Chem. 2011 Feb 25;286(8):6336-44. doi: 10.1074/jbc.M110.154195. Epub 2010 Nov 17.
Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.
糖尿病组织富含一种“激活”形式的人醛糖还原酶（hAR），这是一种参与糖代谢的 NADPH 依赖性氧化还原酶。激活的 hAR 对潜在的抗糖尿病药物的敏感性降低。尽管 hAR 的 C298S 突变体仅通过用氧替换单个硫原子与野生型 hAR 不同，但它复制了激活的 hAR 的许多特征。等温滴定量热法测量显示，NADPH 与 C298S 突变体的结合常数降低了两倍，而 NADP(+)的结合常数保持不变。同样，NADPH 与 C298S 突变体结合的热容变化增加了一倍；然而，NADP(+)与 C298S 结合的热容变化几乎没有差异。野生型和 C298S hAR 的 X 射线晶体结构表明，残基 298 的侧链形成了到烟酰胺口袋的门，并且与丝氨酸相比，半胱氨酸更具柔韧性。与 Cys-298 不同，Ser-298 与 Tyr-209 形成跨越烟酰胺环的氢键，从而抑制烟酰胺的运动。我们假设氧化的烟酰胺的增加极性削弱了 Ser-298 可能形成的氢键，因此，解释了突变对 NADP(+)结合的相对较小的影响。该突变对各种底物的催化速率常数和结合常数的影响与激活的 hAR 相同。因此，进一步证实激活的 hAR 源自 Cys-298 的氧化修饰，Cys-298 是靠近烟酰胺结合口袋的残基。
