Nowak T
J Biol Chem. 1976 Jan 10;251(1):73-8.
The interaction of a series of alkylamines with muscle pyruvate kinase was investigated by kinetic and physical studies in order to understand the mechanisms by which certain monovalent cations can activate the enzyme and to define several of the important conformational changes necessary for catalytic activity. Monomethylammonium ion interacts with pyruvate kinase to activate the enzyme. Dimethyland trimethylammonium ions do not activate, but are competitive inhibitors against activating cations. Tetramethylammonium ion neither activates nor inhibits pyruvate kinase activity. When the enzyme is in the presence of monomethylammonium ion or dimethylammonium ion, a conformational change is observed by ultraviolet difference spectroscopy. This conformational change is similar to that observed with other activating cations and appears to be a necessary but no sufficient conformational change in the formation of an active complex. The interaction of the substrate phosphoenolpyruvate with the pyruvate kinase-Mn2+ complex in the presence of these cations was studied by water proton relaxation rate measurements. The affinity of the enzyme-Mn2+ complex for phosphoenolpyruvate is decreased by a factor of 5 in the presence of any of the alkylamines compared to the affinity measured in the presence of K+ or NH4+. No change in the Km of phosphoenolpyruvate is observed however when it is measured in the presence of monomethylammonium ion, suggesting that the decrease in affinity for the substrate is not the reason for lack of enzymic activity. The conformation of the ternary enzyme-Mn2+-phosphoenolpyruvate complex about the bound Mn2+, as reflected by the enhancement values (epsilont) measured, differs depending upon the nature of the monovalent cation. The epsilon t values measured in the presence of the alkylamines are larger (epsilont - 5.7 +/- 0.2) than those measured in the presence of K+ or NH4+ (epsilont = 1.9 +/- 0.1).
通过动力学和物理研究，对一系列烷基胺与肌肉丙酮酸激酶的相互作用进行了研究，以了解某些单价阳离子激活该酶的机制，并确定催化活性所需的几个重要构象变化。一甲铵离子与丙酮酸激酶相互作用以激活该酶。二甲铵离子和三甲铵离子不激活该酶，但对激活阳离子具有竞争性抑制作用。四甲铵离子既不激活也不抑制丙酮酸激酶活性。当酶存在于一甲铵离子或二甲铵离子中时，通过紫外差光谱法观察到构象变化。这种构象变化与其他激活阳离子所观察到的相似，似乎是形成活性复合物时必要但不充分的构象变化。在这些阳离子存在下，通过水质子弛豫速率测量研究了底物磷酸烯醇丙酮酸与丙酮酸激酶-Mn2+复合物的相互作用。与在K+或NH4+存在下测得的亲和力相比，在任何一种烷基胺存在下，酶-Mn2+复合物对磷酸烯醇丙酮酸的亲和力降低了5倍。然而，当在一甲铵离子存在下测量时，未观察到磷酸烯醇丙酮酸的Km有变化，这表明对底物亲和力的降低不是酶活性缺乏的原因。根据测得的增强值（εt）反映，三元酶-Mn2+-磷酸烯醇丙酮酸复合物围绕结合的Mn2+的构象因单价阳离子的性质而异。在烷基胺存在下测得的εt值（εt = 5.7 +/- 0.2）比在K+或NH4+存在下测得的（εt = 1.9 +/- 0.1）大。
