Identification of essential histidine residues in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase: analysis by chemical modification with diethyl pyrocarbonate and site-directed mutagenesis.
Identification of essential histidine residues in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase: analysis by chemical modification with diethyl pyrocarbonate and site-directed mutagenesis.
复制标题
3-脱氧-D-甘露辛糖酸 8-磷酸合酶中必需组氨酸残基的鉴定：通过焦碳酸二乙酯化学修饰和定点诱变进行分析。
DOI：
10.1021/bi9829884
复制
发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Woodard,RW
中科院分区：
文献类型：
--
作者：
Sheflyan,GY;Duewel,HS;Chen,G;Woodard,RW
来自大肠杆菌的3-脱氧-d-甘露-辛酮糖酸8-磷酸（KDO 8-P）合酶催化d-阿拉伯糖5-磷酸（A5-P）和磷酸烯醇丙酮酸（PEP）的醛醇型缩合生成KDO 8-P和无机磷酸（Pi），该酶被焦碳酸二乙酯（DEPC）灭活。酶和DEPC的失活是一级的。在pH7.6和4 °C时，二级反应速率常数为340 M ~（-1）min ~（-1）。失活速率取决于pH值，pH值失活速率数据表明涉及pKa值为7.3的氨基酸残基。用羟胺处理后，KDO 8-P合酶活性没有恢复到DEPC失活的酶。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基化有关。KDO 8-P合酶被PEP保护免于DEPC失活，并被A5-P部分保护免于失活。为了提供组氨酸残基在由KDO 8-P合酶催化的醛醇型缩合中的参与或作用的进一步证据，将所有六个组氨酸单独突变为甘氨酸或丙氨酸。与野生型酶相比，三种突变体H40 A、H67 G和H246 G的动力学常数不受影响。相比之下，H241 G显示PEP和A5-P的Km增加>10倍，并且降低4倍，而H97 G显示仅A5-P的Km增加，并且降低2倍。H202 G突变体的活性太低而不能精确测量，但获得的数据表明约400倍的降低。野生型和突变体酶的圆二色性测量表明，只有在完全活跃的H67 G和H246 G突变体适度的结构变化。H241 G突变体的保护DEPC失活PEP和A 5-P的野生型酶的相同程度，这表明功能上重要的H241可能不位于附近的底物结合位点。H97 G突变体受PEP保护，使其免于DEPC失活，其程度与野生型酶相同，但不再受A5-P保护。在H202 G突变体的情况下，A5-P和PEP均保护突变体免于DEPC失活，但与野生型酶所观察到的程度不同。在咪唑存在下，H97 G突变体的催化活性部分恢复（野生型活性的20% → 60%），而在咪唑存在下，H202 G突变体的活性恢复较少（野生型活性的<1% → 4%）。
The enzyme 3-deoxy-d-manno-octulosonic acid 8-phosphate (KDO 8-P) synthase fromEscherichia colithat catalyzes the aldol-type condensation ofd-arabinose 5-phosphate (A 5-P) and phosphoenolpyruvate (PEP) to give KDO 8-P and inorganic phosphate (Pi) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first-order in enzyme and DEPC. A second-order rate constant of 340 M-1min-1is obtained at pH 7.6 and 4 °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 pKavalue 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 inKMfor both PEP and A 5-P and a 4-fold reduction inkcat, while H97G demonstrates an increase inKMfor only A 5-P and a 2-fold reduction inkcat. The activity of the H202G mutant was too low to be measured accurately but the data obtained indicated an approximate 400-fold reduction inkcat. 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.