PHOSPHONATE ANALOGS OF PYRUVATE - PROBES OF SUBSTRATE BINDING TO PYRUVATE OXIDASE AND OTHER THIAMIN PYROPHOSPHATE-DEPENDENT DECARBOXYLASES
PHOSPHONATE ANALOGS OF PYRUVATE - PROBES OF SUBSTRATE BINDING TO PYRUVATE OXIDASE AND OTHER THIAMIN PYROPHOSPHATE-DEPENDENT DECARBOXYLASES
复制标题
DOI：
10.1016/0005-2744(80)90186-2
复制
发表时间：
1980-01-01
期刊：
影响因子：
--
通讯作者：
GENNIS, RB
中科院分区：
文献类型：
--
作者：
OBRIEN, TA;KLUGER, R;GENNIS, RB
许多酶通过辅酶硫胺素焦磷酸盐的共价参与催化从丙酮酸中去除二氧化碳。脱羧加合物羟乙基硫胺素焦磷酸向后续产物的转化使这些酶的功能与众不同。乙醛由丙酮酸脱羧酶产生，乙酸由丙酮酸氧化酶产生，乙酰辅酶A由丙酮酸脱氢酶产生。羟乙基硫胺素焦磷酸分解之前步骤的差异和细节可以通过使用两种底物类似物（乙酰膦酸甲酯和乙酰膦酸）进行评估。乙酰膦酸甲酯和乙酰膦酸与大肠杆菌丙酮酸氧化酶和大肠杆菌丙酮酸脱氢酶是丙酮酸的竞争性抑制剂，但氧化酶的 Ki 值比脱氢酶高 3 个数量级以上。在相同条件下，酵母丙酮酸脱羧酶根本不受抑制。乙酰膦酸甲酯的结合导致配体诱导的氧化酶近紫外圆二色光谱的变化。这种光谱扰动仅在辅因子焦磷酸硫胺素存在时出现，强烈表明抑制剂与酶上的底物丙酮酸结合在同一位点。动力学数据表明丙酮酸氧化酶的脂质激活剂明显增加了该酶对丙酮酸及其类似物的亲和力。
A number of enzymes catalyze the removal of CO2 from pyruvate through covalent participation of the coenzyme thiamin pyrophospate. The conversions of the decarboxylated adduct, hydroxyethyl thiamin pyrophosphate, to subsequent products distinguishes the function of these enzymes. Acetaldehyde is produced by pyruvate decarboxylase, acetic acid by pyruvate oxidase and acetyl CoA by pyruvate dehydrogenase. Differences and details of steps prior to decomposition of hydroxyethyl thiamin pyrophosphate can be evaluated through the use of 2 substrate analogues, methyl acetylphosphonate and acetylphosphonate. Methyl acetylphosphonate and acetylphosphonate are competitive inhibitors toward pyruvate with Escherichia coli pyruvate oxidase and E. coli pyruvate dehydrogenase but the value of the Ki for the oxidase is more than 3 orders of magnitude higher than for the dehydrogenase. Yeast pyruvate decarboxylase is not inhibited at all under the same conditions. The binding of methyl acetylphosphonate results in ligand-induced changes in the near UV circular dichroism spectrum of the oxidase. This spectral perturbation is only seen in the presence of the cofactor, thiamin pyrophosphate, strongly suggesting that the inhibitor is binding at the same site as the substrate, pyruvate, on the enzyme. Kinetic data suggest that lipid activators of pyruvate oxidase apparently increase the affinity of the enzyme for pyruvate and its analogues.