Allen K N, Lavie A, Farber G K, Glasfeld A, Petsko G A, Ringe D
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110.
Biochemistry. 1994 Feb 15;33(6):1481-7. doi: 10.1021/bi00172a026.
The D-xylose isomerase of Streptomyces olivochromogenes is a Mg2+- or Mn(2+)-dependent enzyme that catalyzes the aldose-ketose isomerization of xylose to xylulose or of glucose to fructose. Proton exchange into water during enzyme-catalyzed isomerization of C-2 tritiated glucose at 15, 25 and 55 degrees C shows < 0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of pH had no effect on the amount of exchange detected. Such a low percentage of exchange is inconsistent with a proton-transfer mechanism as the main kinetic pathway for isomerization. 19F NMR experiments showed no release of fluoride after incubation of the enzyme for 4 weeks with 800 mM 3-deoxy-3-fluoroglucose or 3-deoxy-3-fluoroallose (both are competitive inhibitors with Ki values of 600 mM). This result is also inconsistent with a proton-transfer mechanism. A hydride-shift mechanism following ring opening has been proposed for the isomerization. Enzyme-catalyzed ring opening was directly measured by demonstrating H2S release upon reaction of xylose isomerase with 1-thioglucose. D-Xylose isomerase-catalyzed interconversion of glucose to fructose exhibited linear Arrhenius behavior with an activation energy of 14 kcal/mol from 0 to 50 degrees C. No change in rate-determining step occurs over this temperature range. 13C NMR experiments with glucose show that enzyme-bound magnesium or manganese does not interact specifically with any one site on the sugar. These results are consistent with nonproductive binding modes for the substrate glucose in addition to productive binding.
橄榄色链霉菌的D-木糖异构酶是一种依赖Mg2+或Mn(2+)的酶，可催化木糖向木酮糖或葡萄糖向果糖的醛糖-酮糖异构化反应。在15、25和55℃下，C-2位含氚葡萄糖在酶催化异构化过程中向水中的质子交换率小于0.6%（即每十亿次周转损失一个质子）。高浓度盐酸胍和极端pH值对检测到的交换量没有影响。如此低的交换百分比与质子转移机制作为异构化的主要动力学途径不一致。19F NMR实验表明，该酶与800 mM 3-脱氧-3-氟葡萄糖或3-脱氧-3-氟阿洛糖（两者均为竞争性抑制剂，Ki值为600 mM）孵育4周后，未检测到氟化物释放。该结果也与质子转移机制不一致。有人提出异构化反应是在开环后发生氢化物转移的机制。通过证明木糖异构酶与1-硫代葡萄糖反应时释放H2S，直接测定了酶催化的开环反应。D-木糖异构酶催化的葡萄糖与果糖的相互转化在0至50℃范围内呈现线性阿伦尼乌斯行为，活化能为14 kcal/mol。在该温度范围内，速率决定步骤没有变化。用葡萄糖进行的13C NMR实验表明，与酶结合的镁或锰不会与糖上的任何一个位点发生特异性相互作用。这些结果与底物葡萄糖除了有生产性结合外还存在非生产性结合模式相一致。
