INTERACTION OF ISOCITRATE DEHYDROGENASE WITH (RS)-3-BROMO-2-KETOGLUTARATE - A POTENTIAL AFFINITY LABEL FOR ALPHA-KETOGLUTARATE BINDING-SITES
INTERACTION OF ISOCITRATE DEHYDROGENASE WITH (RS)-3-BROMO-2-KETOGLUTARATE - A POTENTIAL AFFINITY LABEL FOR ALPHA-KETOGLUTARATE BINDING-SITES
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DOI：
10.1021/bi00507a037
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发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
HARTMAN, FC
中科院分区：
文献类型：
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作者：
HARTMAN, FC
研究了氧化的尼古丁腺嘌呤二核苷酸磷酸依赖异柠檬酸脱氢酶（来自猪心脏）与(RS)-3-溴-2-酮戊二酸的相互作用，以评价该试剂作为. α选择性试剂的潜力。-酮戊二酸结合位点。用0.1 mM溴酮戊二酸盐在pH 7.4下使酶迅速失活。随着试剂浓度的增加，反应呈现速率饱和；最小失活半衰期为3分钟，溴酮戊二酸盐的Kinact[失活常数]为250 μ m。异柠檬酸盐和NADP+可以防止失活，而酮戊二酸则没有。在监测异柠檬酸氧化的试验中，溴酮戊二酸盐是脱氢酶的竞争性抑制剂（Ki = 100 mM）。通过NADPH的氧化判断，溴酮戊二酸也是异柠檬酸脱氢酶的底物，面积为250亩。M和Vmax与异柠檬酸氧化相当。异柠檬酸盐（Ki = 3 .mu）竞争性地抑制溴酮戊二酸的还原。M)和戊二酸酮（Ki = 50 μ M）。与酶催化氧化异柠檬酸盐一样，溴酮戊二酸盐的还原具有立体特异性，需要二价金属离子，对NADPH具有绝对特异性。由于溴酮戊二酸的催化转化不需要二氧化碳，其还原可能与草酰琥珀酸的还原相当，而不是酮戊二酸的还原羧化。虽然溴酮戊二酸盐作为异柠檬酸脱氢酶的底物，显然对活性位点具有亲和力，但该试剂对酶的不可逆失活可能是由于活性位点区域以外的修饰，因为溴酮戊二酸盐在催化转化过程中未观察到失活。用亲和层析法对溴酮戊二酸烷基化的巯基琼脂糖进行了12倍纯化。
The interaction of oxidized nicotine adenine dinucleotide phosphate dependent isocitrate dehydrogenase (from pig heart) with (RS)-3-bromo-2-ketoglutarate was investigated in an effort to evaluate the reagent''s potential as a selective reagent for .alpha.-ketoglutarate binding sites. The enzyme is rapidly inactivated by 0.1 mM bromoketoglutarate at pH 7.4. With increasing concentrations of reagent, the reaction shows a rate saturation; the minimum inactivation half-time is 3 min and the Kinact [inactivation constant] for bromoketoglutarate is 250 .mu.M. Isocitrate and NADP+ protect against inactivation, while ketoglutarate does not. When tested in the assay that monitors isocitrate oxidation, bromoketoglutarate is a competitive inhibitor (Ki = 100 mM) of the dehydrogenase. As judged by oxidation of NADPH, bromoketoglutarate is also a substrate for isocitrate dehydrogenase, exhibiting a Km of 250 .mu.M and a Vmax comparable to that for isocitrate oxidation. The reduction of bromoketoglutarate is competitively inhibited by isocitrate (Ki = 3 .mu.M) and ketoglutarate (Ki = 50 .mu.M). Like the enzyme-catalyzed oxidation of isocitrate, the reduction of bromoketoglutarate is stereospecific, requires divalent metal ions and shows absolute specificity for NADPH. Since CO2 is not required for catalytic turnover of bromoketoglutarate, its reduction is likely comparable to that of oxalosuccinate rather than the reductive carboxylation of ketoglutarate. Although bromoketoglutarate, as a substrate for isocitrate dehydrogenase, clearly has affinity for the active site, the irreversible inactivation of the enzyme by the reagent may result from modification outside the active-site region, since inactivation during catalytic turnover of bromoketoglutarate is not observed. Commercial isocitrate dehydrogenase is purified 12-fold by affinity chromatography on thiol-agarose alkylated by bromoketoglutarate.