Bloxham D P, Chalkley R A
Biochem J. 1976 Nov;159(2):201-11. doi: 10.1042/bj1590201.
Rabbit muscle pyruvate kinase was irreverisbly inactivated by 5-chloro-4-oxopentanoic acid with a pKa of 9.2. The inhibition was time-dependent and was related to the 5-chloro-4-oxopentanoic acid concentration. Analysis of the kinetics of inhibition showed that the binding of the inhibitor showed positive co-operativity (n = 1.5 +/- 0.2). Inhibition of pyruvate kinase by 5-chloro-4-oxopentanoic acid was prevented by ligands which bind to the active site. Their effectiveness was placed in the order Mg2+ greater than phosphoenolpyruvate greater than ATP greater than ADP greater than pyruvate. Inhibitor-modified pyruvate kinase was unable to catalyse the detritiation of [3-(3)H]pyruvate in the ATP-promoted reaction, but it did retain 5-10% of the activity with either phosphate or arsenate as promoters. 5-Chlor-4-oxo-[3,5-(3)H]pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoicheiometry of 1 mol of inhibitor bound per mol of pyruvate kinase protomer. The incorporation of the inhibitor and the loss of enzyme was proportional. These results are discussed in terms of 5-chloro-4-oxopentanoic acid alkylating a functional group in the phosphoryl overlap region of the active site, and a model is presented in which this compound alkylates an active-site thiol in a reaction that is controlled by a more basic group at the active site.
兔肌肉丙酮酸激酶可被pKa为9.2的5-氯-4-氧代戊酸不可逆地失活。抑制作用具有时间依赖性，且与5-氯-4-氧代戊酸浓度相关。抑制动力学分析表明，抑制剂的结合表现出正协同性（n = 1.5±0.2）。与活性位点结合的配体可阻止5-氯-4-氧代戊酸对丙酮酸激酶的抑制。它们的有效性顺序为：Mg2+＞磷酸烯醇丙酮酸＞ATP＞ADP＞丙酮酸。抑制剂修饰的丙酮酸激酶在ATP促进的反应中无法催化[3-(3)H]丙酮酸的脱氚反应，但以磷酸盐或砷酸盐作为促进剂时，它仍保留5 - 10%的活性。5-氯-4-氧代-[3,5-(3)H]戊酸与丙酮酸激酶共价结合，表明每摩尔丙酮酸激酶原体结合1摩尔抑制剂，抑制剂的掺入与酶活性的丧失成正比。本文根据5-氯-4-氧代戊酸使活性位点磷酰重叠区域的一个官能团烷基化来讨论这些结果，并提出了一个模型，其中该化合物在由活性位点一个碱性更强的基团控制的反应中使活性位点的一个硫醇烷基化。
