Podestá F E, Plaxton W C
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Biochim Biophys Acta. 1992 Nov 20;1160(2):213-20. doi: 10.1016/0167-4838(92)90010-b.
The kinetic properties of cytosolic pyruvate kinase (PKc) from germinating castor oil seeds (COS) have been investigated. From experiments in which the free Mg2+ concentration was varied at constant levels of either the complexed or free forms of the substrates it was determined that the true substrates are the free forms of both phosphoenolpyruvate (PEP) and ADP. This conclusion is corroborated by the quenching of intrinsic PKC tryptophan fluorescence by free PEP and ADP. Mg2+ is bound as the free bivalent cation but is likely released as MgATP. The fluorescence data, substrate interaction kinetics, and pattern of inhibition by products and substrate analogues (adenosine 5'-O-(2-thiodiphosphate) for ADP and phenyl phosphate for PEP) are compatible with a sequential, compulsory-ordered, Tri-Bi type kinetic reaction mechanism. PEP is the leading substrate, and pyruvate the last product to abandon the enzyme. The dissociation constant and limiting Km for free PEP (8.2 to 22 and 38 microM, respectively) and the limiting Km for free ADP (2.9 microM) are considerably lower than those reported for the non-plant enzyme. The results indicate that COS PKc exists naturally in an activated state, similar to the fructose 1,6-bisphosphate-activated yeast enzyme. This deduction is consistent with a previous study (F.E. Podestá and W.C. Plaxton (1991) Biochem. J. 279, 495-501) that failed to identify any allosteric activators for the COS PKc, but which proposed a regulatory mechanism based upon ATP levels and pH-dependent alterations in the enzyme's response to various metabolite inhibitors. As plant phosphofructokinases display potent inhibition by PEP, the overall rate of glycolytic flux from hexose 6-phosphate to pyruvate in the plant cytosol will ultimately depend upon variations in PEP levels brought about by the regulation of PKc.
对蓖麻籽（COS）萌发过程中胞质丙酮酸激酶（PKc）的动力学特性进行了研究。在实验中，当底物的络合形式或游离形式处于恒定水平时，改变游离Mg2+浓度，结果确定真正的底物是磷酸烯醇式丙酮酸（PEP）和ADP的游离形式。游离PEP和ADP对PKC内在色氨酸荧光的猝灭证实了这一结论。Mg2+以游离二价阳离子的形式结合，但可能以MgATP的形式释放。荧光数据、底物相互作用动力学以及产物和底物类似物（ADP的腺苷5'-O-(2-硫代二磷酸)和PEP的苯磷酸）的抑制模式与顺序、强制有序的Tri-Bi型动力学反应机制相符。PEP是主要底物，丙酮酸是最后离开酶的产物。游离PEP的解离常数和极限Km（分别为8.2至22 μM和38 μM）以及游离ADP的极限Km（2.9 μM）远低于非植物酶的报道值。结果表明，COS PKc天然以活化状态存在，类似于果糖1,6-二磷酸激活的酵母酶。这一推论与先前的一项研究（F.E. Podestá和W.C. Plaxton（1991年）《生物化学杂志》279卷，495 - 501页）一致，该研究未能鉴定出COS PKc的任何变构激活剂，但提出了一种基于ATP水平和pH依赖性改变酶对各种代谢物抑制剂反应的调节机制。由于植物磷酸果糖激酶受到PEP的强烈抑制，植物细胞质中从6-磷酸己糖到丙酮酸的糖酵解通量的总体速率最终将取决于PKc调节引起的PEP水平变化。
