Kubiseski T J, Flynn T G
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
J Biol Chem. 1995 Jul 14;270(28):16911-7.
Aldose reductase (ALR2) shows a strong specificity for its nucleotide coenzyme, binding NADPH much more tightly than NADH (KD of < 1 microM versus 1.2 mM respectively). Interactions responsible for this specificity include salt linkages between the highly conserved residues Lys-262 and Arg-268, and the 2'-phosphate of NADP(H). Previous studies show that mutation of Lys-262 results in an increase in the Km for both coenzyme and aldehyde substrate, as well as in the kcat of reduction. The present study shows that mutation of Arg-268 to methionine results in a 36-fold increase in Km and 205-fold increase in KD for NADPH, but little change in Km for DL-glyceraldehyde or in the kcat of the reaction. Calculation of free energy changes show that the 2'-phosphate of NADPH contributes 4.7 kcal/mol of binding energy to its interaction with WT-hALR2. For the R268M mutant, the interaction of NADPH was destabilized by 3.2 kcal/mol, indicating that the mutation decreases the binding energy of NADPH by 65%. The effect of removing Arg-268 in the absence of the 2'-phosphate of NADPH was virtually identical to the destabilization of the activation energy in the absence of the 2'-phosphate itself (1.9 versus 2.0 kcal/mol, respectively). Therefore, while the 2'-phosphate of the coenzyme plays a role in both coenzyme binding and transition state stabilization during catalysis, the role of Arg-268 lies strictly in tighter coenzyme binding.
醛糖还原酶（ALR2）对其核苷酸辅酶表现出很强的特异性，与NADPH的结合比与NADH紧密得多（解离常数分别为<1微摩尔与1.2毫摩尔）。造成这种特异性的相互作用包括高度保守的残基Lys-262和Arg-268之间的盐键，以及NADP(H)的2'-磷酸基团。先前的研究表明，Lys-262的突变导致辅酶和醛底物的米氏常数增加，以及还原反应的催化常数增加。本研究表明，Arg-268突变为甲硫氨酸会导致NADPH的米氏常数增加36倍，解离常数增加205倍，但对DL-甘油醛的米氏常数或反应的催化常数影响很小。自由能变化的计算表明，NADPH的2'-磷酸基团在与野生型人醛糖还原酶（WT-hALR2）的相互作用中贡献了4.7千卡/摩尔的结合能。对于R268M突变体，NADPH的相互作用不稳定了3.2千卡/摩尔，表明该突变使NADPH的结合能降低了65%。在没有NADPH的2'-磷酸基团的情况下去除Arg-268的效果与在没有2'-磷酸基团本身的情况下活化能的不稳定效果几乎相同（分别为1.9千卡/摩尔与2.0千卡/摩尔）。因此，虽然辅酶的2'-磷酸基团在催化过程中的辅酶结合和过渡态稳定中都起作用，但Arg-268的作用严格限于更紧密的辅酶结合。
