Sheflyan G Y, Duewel H S, Chen G, Woodard R W
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA.
Biochemistry. 1999 Oct 26;38(43):14320-9. doi: 10.1021/bi9829884.
The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) synthase from Escherichia coli that catalyzes the aldol-type condensation of D-arabinose 5-phosphate (A 5-P) and phosphoenolpyruvate (PEP) to give KDO 8-P and inorganic phosphate (P(i)) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first-order in enzyme and DEPC. A second-order rate constant of 340 M(-1) min(-1) is obtained at pH 7.6 and 4 degrees C. The rate of inactivation is dependent on pH and the pH-inactivation rate data imply the involvement of an amino acid residue with a pK(a) value of 7.3. KDO 8-P synthase activity is not restored to the DEPC-inactivated enzyme following treatment with hydroxylamine. Complete loss of KDO 8-P synthase activity correlates with the ethoxyformylation of three histidine residues by DEPC. KDO 8-P synthase is protected against DEPC inactivation by PEP and partially protected against inactivation by A 5-P. To provide further evidence for the involvement or role of the histidine residues in the aldol-type condensation catalyzed by KDO 8-P synthase, all six histidines were individually mutated to either glycine or alanine. The kinetic constants for the three mutants H40A, H67G, and H246G were unaffected as compared to the wild type enzyme. In contrast, H241G demonstrates a >10-fold increase in K(M) for both PEP and A 5-P and a 4-fold reduction in k(cat), while H97G demonstrates an increase in K(M) for only A 5-P and a 2-fold reduction in k(cat). The activity of the H202G mutant was too low to be measured accurately but the data obtained indicated an approximate 400-fold reduction in k(cat). Circular dichroism measurements of the wild-type and mutant enzymes indicate modest structural changes in only the fully active H67G and H246G mutants. The H241G mutant is protected against DEPC inactivation by PEP and A 5-P to the same extent as the wild-type enzyme, suggesting that the functionally important H241 may not be located in the vicinity of the substrate binding sites. The H97G mutant is protected by PEP against DEPC inactivation to the same degree as the wild-type enzyme but is no longer protected by A 5-P. In the case of the H202G mutant, both A 5-P and PEP protect the mutant against DEPC inactivation but to different extents from those observed for the wild-type enzyme. The catalytic activity of the H97G mutant is partially restored (20% --> 60% of wild-type activity) in the presence of imidazole, while a minor amount of activity is restored to the H202G mutant (<1% --> 4% of wild-type activity) in the presence of imidazole.
来自大肠杆菌的3-脱氧-D-甘露糖辛酮酸8-磷酸（KDO 8-P）合酶催化5-磷酸-D-阿拉伯糖（A 5-P）和磷酸烯醇丙酮酸（PEP）的醛醇型缩合反应生成KDO 8-P和无机磷酸（P(i)），该酶会被焦碳酸二乙酯（DEPC）灭活。这种灭活反应对酶和DEPC而言是一级反应。在pH 7.6和4℃条件下，获得的二级速率常数为340 M⁻¹ min⁻¹。灭活速率取决于pH，且pH-灭活速率数据表明存在一个pK(a)值为7.3的氨基酸残基参与其中。用羟胺处理后，KDO 8-P合酶活性无法恢复到被DEPC灭活的酶。KDO 8-P合酶活性的完全丧失与DEPC对三个组氨酸残基的乙氧甲酰化作用相关。PEP可保护KDO 8-P合酶免受DEPC灭活，A 5-P则提供部分保护。为进一步证明组氨酸残基在KDO 8-P合酶催化的醛醇型缩合反应中的参与情况或作用，将所有六个组氨酸分别突变为甘氨酸或丙氨酸。与野生型酶相比，三个突变体H40A、H67G和H246G的动力学常数未受影响。相比之下，H241G对PEP和A 5-P的K(M)均增加了10倍以上，k(cat)降低了4倍，而H97G仅对A 5-P的K(M)增加，k(cat)降低了2倍。H202G突变体的活性过低，无法准确测量，但获得的数据表明k(cat)大约降低了400倍。野生型和突变型酶的圆二色性测量表明，只有完全活性的H67G和H246G突变体有适度的结构变化。H241G突变体与野生型酶一样，受到PEP和A 5-P对DEPC灭活的保护，这表明功能上重要的H241可能不在底物结合位点附近。H97G突变体受到PEP对DEPC灭活的保护程度与野生型酶相同，但不再受到A 5-P的保护。对于H202G突变体，A 5-P和PEP均保护该突变体免受DEPC灭活，但程度与野生型酶不同。在咪唑存在的情况下，H97G突变体的催化活性部分恢复（从野生型活性的20%恢复到60%），而在咪唑存在的情况下，H202G突变体恢复了少量活性（从野生型活性的<1%恢复到4%）。
