Erythrocyte pyruvate kinase deficiency. The influence of physiologically important metabolites on the function of normal and defective enzymes.

Enzyme & protein Pub Date : 1994-01-01
M Lakomek, H Winkler, A Pekrun, N Krüger, M Sander, P Huppke, W Schröter
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Abstract

The dependence of the erythrocyte pyruvate kinase (PK)-catalyzed reaction on the glycolytic intermediates glucose-6-phosphate (Gluc-6-P), 2,3-diphosphoglycerate (2,3-DPG) and the nucleotides ADP and ATP was studied in normal individuals and 14 patients with PK deficiency. The Gluc-6-P concentrations in the erythrocytes are markedly elevated (4- to 6-fold) in 9 patients with severe hemolytic anemia compared to those 5 exhibiting a mild clinical course (up to 2-fold increased). 2,3-DPG is elevated up to 2 times compared to the controls whereas the measured ADP and ATP only slightly deviate from the normal range. Control experiments showed that these elevations of Gluc-6-P and 2,3-DPG do not depend on the number of reticulocytes. In enzyme kinetic terms, Gluc-6-P shifts the Hill coefficient to smaller values, i.e. suppresses the positive cooperativity (sigmoidal reaction kinetics), found in normal and some of the mutant enzymes and shift the noncooperative enzymes of some patients to an enzyme exhibiting negative cooperativity. The negative cooperativity already present in the enzymes of some of the patients suffering from severe hemolytic anemia becomes more pronounced upon addition of Gluc-6-P. Apparently 2,3-DPG acts as an antagonist to Gluc-6-P in increasing the Hill coefficient, i.e. enhancing the positive cooperativity of the normal enzyme. It shifts the hyperbolic patients' enzymes to a sigmoidal reaction type and the enzymes of those patients with negative cooperativity to a hyperbolic type. ADP and ATP show a similar behavior as 2,3-DPG, but additionally inhibit the enzyme at higher concentrations. The influence of all four phosphates on the Michaelis constant varies depending on the type of cooperativity, in some cases increasing and in some cases decreasing K0.5 PEP. With 7 of the patients, all of them with severe clinical course, a genetic analysis of their R-type PK gene was performed and genetic defects have been identified in the coding sequence. The found changes in the amino acid sequence and their corresponding location in the tertiary structure of the PK subunit can satisfactorily explain the alterations of the regulatory properties of the mutant enzymes thus allowing to establish a good correlation between altered structural and functional properties of the deficient enzyme and the severeness of the course of the disease.

红细胞丙酮酸激酶缺乏。生理上重要的代谢物对正常和缺陷酶功能的影响。
研究了红细胞丙酮酸激酶(PK)催化反应对糖酵解中间体葡萄糖-6-磷酸(葡萄糖-6-p)、2,3-二磷酸甘油酸(2,3- dpg)及核苷酸ADP和ATP的依赖性。在9例严重溶血性贫血患者中,红细胞中葡萄糖-6- p浓度明显升高(4- 6倍),而在5例表现出轻度临床病程的患者中(升高高达2倍)。与对照组相比,2,3- dpg升高高达2倍,而测量的ADP和ATP仅略微偏离正常范围。对照实验表明,葡萄糖-6- p和2,3- dpg的升高不依赖于网状细胞的数量。在酶动力学方面,葡萄糖-6- p将希尔系数移至较小的值,即抑制正常和某些突变酶中的正协同性(s型反应动力学),并将一些患者的非协同性酶转移到表现负协同性的酶上。在一些患有严重溶血性贫血的患者的酶中已经存在的负协同性在加入葡萄糖-6- p后变得更加明显。显然,2,3- dpg可以作为葡萄糖-6- p的拮抗剂,增加Hill系数,即增强正常酶的正协同性。它将双曲型患者的酶转化为s型反应型,将负协同性患者的酶转化为双曲型反应型。ADP和ATP表现出与2,3- dpg相似的行为,但在较高浓度下会对酶产生抑制作用。所有四种磷酸盐对米切里斯常数的影响取决于协同作用的类型,在某些情况下增加K0.5 PEP,在某些情况下减少K0.5 PEP。其中7例患者均有严重的临床病程,对其r型PK基因进行了遗传分析,并在编码序列中发现遗传缺陷。所发现的氨基酸序列的变化及其在PK亚基三级结构中相应位置的变化可以令人满意地解释突变酶的调节特性的改变,从而使缺陷酶的结构和功能特性的改变与疾病的严重程度建立良好的相关性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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