Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000, USA.
Biochemistry. 2011 Nov 22;50(46):10170-81. doi: 10.1021/bi201378c. Epub 2011 Oct 27.
D-Xylose isomerase (XI) and triosephosphate isomerase (TIM) catalyze the aldose-ketose isomerization reactions of D-xylose and d-glyceraldehyde 3-phosphate (DGAP), respectively. D-Glyceraldehyde (DGA) is the triose fragment common to the substrates for XI and TIM. The XI-catalyzed isomerization of DGA to give dihydroxyacetone (DHA) in D(2)O was monitored by (1)H nuclear magnetic resonance spectroscopy, and a k(cat)/K(m) of 0.034 M(-1) s(-1) was determined for this isomerization at pD 7.0. This is similar to the k(cat)/K(m) of 0.017 M(-1) s(-1) for the TIM-catalyzed carbon deprotonation reaction of DGA in D(2)O at pD 7.0 [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. The much larger activation barrier for XI-catalyzed isomerization of D-xylose (k(cat)/K(m) = 490 M(-1) s(-1)) versus that for the TIM-catalyzed isomerization of DGAP (k(cat)/K(m) = 9.6 × 10(6) M(-1) s(-1)) is due to (i) the barrier to conversion of cyclic d-xylose to the reactive linear sugar (5.4 kcal/mol) being larger than that for conversion of DGAP hydrate to the free aldehyde (1.7 kcal/mol) and (ii) the intrinsic binding energy [Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219-410] of the terminal ethylene glycol fragment of D-xylose (9.3 kcal/mol) being smaller than that of the phosphodianion group of DGAP (~12 kcal/mol). The XI-catalyzed isomerization of DGA in D(2)O at pD 7.0 gives a 90% yield of [1-(1)H]DHA and a 10% yield of [1-(2)H]DHA, the product of isomerization with incorporation of deuterium from solvent D(2)O. By comparison, the transfer of (3)H from the labeled hexose substrate to solvent is observed only once in every 10(9) turnovers for the XI-catalyzed isomerization of [2-(3)H]glucose in H(2)O [Allen, K. N., Lavie, A., Farber, G. K., Glasfeld, A., Petsko, G. A., and Ringe, D. (1994) Biochemistry 33, 1481-1487]. We propose that truncation of the terminal ethylene glycol fragment of d-xylose to give DGA results in a large decrease in the rate of XI-catalyzed isomerization with hydride transfer compared with that for proton transfer. An ultra-high-resolution (0.97 Å) X-ray crystal structure was determined for the complex obtained by soaking crystals of XI with 50 mM DGA. The triose binds to XI as the unreactive hydrate, but ligand binding induces metal cofactor movement and conformational changes in active site residues similar to those observed for XI·sugar complexes.
D-木糖异构酶（XI）和磷酸丙糖异构酶（TIM）分别催化 D-木糖和 d-甘油醛 3-磷酸（DGAP）的醛酮糖异构化反应。D-甘油醛（DGA）是 XI 和 TIM 的底物共有的三碳片段。在 D2O 中，通过（1）H 核磁共振光谱监测 XI 催化的 DGA 异构化为二羟丙酮（DHA）的反应，在 pD7.0 下确定了该异构化的 kcat/Km 为 0.034 M-1 s-1。这与 TIM 催化的 D2O 中 DGA 的碳去质子化反应的 kcat/Km（在 pD7.0 下为 0.017 M-1 s-1）相似[Amyes，TL，O'Donoghue，AC 和 Richard，JP（2001）J. Am. Chem. Soc. 123，11325-11326]。XI 催化的 D-木糖异构化的活化能垒（kcat/Km = 490 M-1 s-1）比 TIM 催化的 DGAP 异构化的活化能垒（kcat/Km = 9.6×106 M-1 s-1）大得多，这是由于（i）环状 D-木糖转化为反应性线性糖的转化障碍（5.4 kcal/mol）大于 DGAP 水合物转化为游离醛的转化障碍（1.7 kcal/mol），（ii）D-木糖末端乙烯二醇片段的固有结合能[Jencks，WP（1975）Adv. Enzymol. Relat. Areas Mol. Biol. 43，219-410]（9.3 kcal/mol）小于 DGAP 磷酸二阴离子基团的固有结合能（约 12 kcal/mol）。在 pD7.0 的 D2O 中，XI 催化的 DGA 异构化产生了 90%的[1-（1）H]DHA 和 10%的[1-（2）H]DHA，这是与溶剂 D2O 中氘掺入的异构化产物。相比之下，在 XI 催化的[2-（3）H]葡萄糖在 H2O 中的异构化中，仅观察到标记己糖底物中的（3）H 向溶剂的转移，每 109 次周转中只有一次[Allen，KN，Lavie，A，Farber，GK，Glasfeld，A，Petsko，GA 和 Ringe，D（1994）Biochemistry 33，1481-1487]。我们提出，D-木糖末端乙烯二醇片段的截断导致 XI 催化的异构化与氢化物转移的速率与质子转移相比大大降低。通过将 XI 与 50 mM DGA 一起浸泡晶体来确定获得的复合物的超高分辨率（0.97Å）X 射线晶体结构。三碳糖以非反应性水合物的形式结合到 XI 上，但配体结合诱导金属辅因子运动和活性位点残基的构象变化，类似于 XI·糖复合物中观察到的变化。
