单价和二价阳离子对乳酸链球菌C10丙酮酸激酶活性的影响。

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

作者信息

Crow V L, Pritchard G G

出版信息

Biochim Biophys Acta. 1977 Mar 15;481(1):105-14. doi: 10.1016/0005-2744(77)90142-5.

Abstract

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.

摘要

来自乳酸链球菌C10的丙酮酸激酶（ATP：丙酮酸2-O-磷酸转移酶，EC 2.7.1.40）对单价阳离子和二价阳离子均有绝对需求。NH₄⁺和K⁺以相似浓度呈S形激活该酶（nH = 1.55），而Na⁺和Li⁺只能微弱激活该酶。在所研究的8种二价阳离子中，只有3种（Co²⁺、Mg²⁺和Mn²⁺）能激活该酶。其余5种二价阳离子（Cu²⁺、Zn²⁺、Ca²⁺、Ni²⁺和Ba²⁺）不同程度地抑制Mg²⁺激活的酶。（Cu²⁺在0.1 mM时完全抑制活性，而Ba²⁺作为最不有效的抑制剂，在3.2 mM时导致50%的抑制）。在1 mM果糖1,6-二磷酸（Fru-1,6-P₂）存在的情况下，该酶对三种激活二价阳离子中的每一种都表现出不同的动力学响应。对于Co²⁺、Mn²⁺和Mg²⁺，希尔相互作用系数（nH）分别为1.6、1.7和2.3，达到最大活性50%所需的相应二价阳离子浓度分别为0.9、0.46和0.9 mM。只有以Mn²⁺作为二价阳离子时，在没有Fru-1,6-P₂的情况下才有显著活性。当Mn²⁺取代Mg²⁺时，Fru-1,6-P₂的激活从S形（nH = 2.0）变为双曲线形（nH = 1.0）动力学，达到最大活性50%所需的Fru-1,6-P₂浓度从0.35 mM降至0.015 mM。在1 mM Fru-1,6-P₂存在的情况下，当Mg²⁺被Mn²⁺取代时，磷酸烯醇丙酮酸结合的协同性增加（nH从1.2变为1.8），给出半数最大速度的磷酸烯醇丙酮酸浓度值降低（从0.18 mM磷酸烯醇丙酮酸降至0.015 mM）。当用Mn²⁺取代Mg²⁺时，对ADP的动力学响应没有明显改变。根据二价阳离子是Mg²⁺还是Mn²⁺，pH对磷酸烯醇丙酮酸和Fru-1,6-P₂结合的影响不同。