3,4-DIDEHYDRO-2-KETOGLUTARATE - AN AFFINITY LABEL FOR DIPHOSPHOPYRIDINE NUCLEOTIDE DEPENDENT ISOCITRATE DEHYDROGENASE
3,4-DIDEHYDRO-2-KETOGLUTARATE - AN AFFINITY LABEL FOR DIPHOSPHOPYRIDINE NUCLEOTIDE DEPENDENT ISOCITRATE DEHYDROGENASE
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DOI：
10.1021/bi00258a026
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发表时间：
1982-01-01
期刊：
影响因子：
2.9
通讯作者：
COLMAN, RF
中科院分区：
文献类型：
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作者：
BEDNAR, RA;HARTMAN, FC;COLMAN, RF
2-酮戊二酸类似物3-溴-2-酮戊二酸在缓冲溶液中分解，产生可与亲核试剂反应的化合物。在pH 6.15的50 mM 2-（N-吗啉代）乙磺酸（Mes）缓冲液中，含有2 mM MnSO 4和20%甘油，溴的损失和损失的能力，作为底物的猪心TPN依赖性异柠檬酸脱氢酶发生的半衰期为1.6小时;然而，损失的能力，与谷胱甘肽发生的半衰期为7小时。据推测，HBr的消除产生不饱和化合物3，4-二-异戊-2-酮戊二酸酯，预期其通过C-4的迈克尔型加成来加成亲核试剂。这种分解产物逐渐使猪心依赖于DPN的异柠檬酸脱氢酶失活。随着试剂浓度的增加，反应表现出速率饱和：最小失活半衰期为35 min，Kinact为1.5 mM。底物异柠檬酸盐提供了针对失活的特异性保护，而辅酶DPN和DPNH仅产生弱保护。假定的3，4-二巯基-2-酮戊二酸与酶一起孵育导致变构ADP活化以与失活相同的速率丧失。异柠檬酸盐和ADP都提供了同等的保护，防止活性丧失和ADP活化丧失，这意味着这两个过程可能是由相同的分子事件引起的。由于ADP活化的损失测量了变构化合物对未修饰的催化活性亚基的影响，因此有强有力的证据表明修饰和未修饰的亚基之间存在相互作用。酶活性的完全丧失与apprx的共价掺入相关。1.0 mol试剂/mol平均亚基。3，4-二异丙基-2-酮戊二酸可能作为DPN依赖性异柠檬酸脱氢酶底物结合位点的亲和标记物。掺入的化学计量表明，酶的每个结构上不同的亚基必须被修饰以完全丧失活性。半胱氨酸残基被鉴定为被3-溴-2-酮戊二酸和推定的3，4-二溴-2-酮戊二酸修饰的氨基酸的主要候选物。该半胱氨酸残基可在酶促反应机制中作为一般碱起作用。
The 2-ketoglutarate analog 3-bromo-2-ketoglutarate decomposes in buffered solution to produce a compound which can react with nucleophiles. At pH 6.15 in 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer containing 2 mM MnSO4 and 20% glycerol, both the loss of bromide and the loss of the ability to serve as a substrate for pig heart TPN-dependent isocitrate dehydrogenase occur with a half-time of 1.6 h; however, the loss of the ability to react with glutathione occurs with a half-time of 7h. Presumably, elimination of HBr yields an unsaturated compound, 3,4-di-dehydro-2-ketoglutarate, which would be expected to add nucleophiles by a Michael-type addition at C-4. This decomposition product progressively inactivates pig heart DPN-dependent isocitrate dehydrogenase. With increasing concentrations of the reagent, the reaction exhibits a rate saturation: the minimum inactivation half-time is 35 min with a Kinact of 1.5 mM. Specific protection against inactivation is afforded by the substrate isocitrate, while the coenzymes DPN and DPNH yield only weak protection. Incubation of the presumed 3,4-didehydro-2-ketoglutarate with the enzyme results in a loss of allosteric ADP activation at the same rate as inactivation. Both isocitrate and ADP afford equal protection against loss of activity and loss of ADP activation, implying that both processes may result from the same molecular event. Since the loss of ADP activation measures the effect of the allosteric compound on unmodified catalytically active subunits, there is strong evidence of interaction between modified and unmodified subunits. Complete loss of enzymatic activity correlates with the covalent incorporation of .apprx. 1.0 mol of reagent/mol of average subunit. 3,4-Di-dehydro-2-ketoglutarate probably functions as an affinity label of the substrate binding site of DPN-dependent isocitrate dehydrogenase. The stoichiometry of incorporation suggests that each of the structurally distinct subunits of the enzyme must be modified for complete loss of activity. A cysteine residue was identified as the prime candidate for the amino acid modified by both 3-bromo-2-ketoglutarate and the putative 3,4-didehydro-2-ketoglurate. This cysteine residue may function as a general base in the enzymic reaction mechanism.