Wiksell E, Larsson-Raźnikiewicz M
Department of Chemistry and Molecular Biology, Swedish University of Agricultural Sciences, Uppsala.
J Biol Chem. 1987 Oct 25;262(30):14472-8.
Yeast phosphoglycerate kinase is irreversibly inactivated upon incubation with 5'-[p-(fluorosulfonyl)-benzoyl]-1-N6-ethenoadenosine (5'-FSB epsilon A), an analogue to the nucleotide substrate. Marked protection against inactivation occurs with MgATP, ATP, MgADP, ADP, and 3-phosphoglycerate, suggesting that a part of the catalytic center is modified. The time dependence of the inactivation is characterized by a nonlinear kinetic profile. Curve fitting of various models for ligand binding to the enzyme suggested a two-site model. Modification of one of the sites appears to protect the catalytically essential site from modification. Stoichiometric studies show that the relationship between moles of 5'-FSB epsilon A incorporated per mole of enzyme and the residual enzymatic activity also shows nonlinear behavior. An extrapolated value of 1.5 mol of bound label/mol of enzyme corresponds to complete inactivation. The apparent overall pseudo first-order rate constant for the reaction between phosphoglycerate kinase and 5'-FSB epsilon A, as well as the separate rate constants for the modification, exhibit saturation behavior with respect to the concentration of 5'-FSB epsilon A, indicative of a rapid reversible binding of the reagent to the enzyme prior to modification.
酵母磷酸甘油酸激酶与核苷酸底物类似物5'-[对-(氟磺酰基)-苯甲酰基]-1-N6-乙烯腺苷(5'-FSBεA)温育后会不可逆地失活。MgATP、ATP、MgADP、ADP和3-磷酸甘油酸对失活有显著的保护作用，这表明催化中心的一部分被修饰了。失活的时间依赖性具有非线性动力学特征。对配体与酶结合的各种模型进行曲线拟合，表明是一个双位点模型。其中一个位点的修饰似乎能保护催化必需位点不被修饰。化学计量学研究表明，每摩尔酶结合的5'-FSBεA摩尔数与残余酶活性之间的关系也呈非线性。每摩尔酶结合1.5摩尔标记物的外推值对应于完全失活。磷酸甘油酸激酶与5'-FSBεA反应的表观总伪一级速率常数以及修饰的单独速率常数，相对于5'-FSBεA的浓度呈现饱和行为，这表明该试剂在修饰之前与酶发生了快速可逆的结合。
