Bollenbach T J, Mesecar A D, Nowak T
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Biochemistry. 1999 Jul 13;38(28):9137-45. doi: 10.1021/bi990690n.
Site-directed mutagenesis was used to change Lys 240 of yeast pyruvate kinase (Lys 269 in muscle PK) to Met. K240M has an absolute requirement for FBP for catalysis. K240M is 100- and 1000-fold less active than wild-type YPK in the presence of Mn(2+) and Mg(2+), respectively. Steady-state fluorescence titration data suggest that the substrate PEP binds to K240M with the same affinity as it does to wild-type YPK. The rate of phosphoryl transfer in K240M has been decreased >1000-fold compared to wild-type YPK. The detritiation of 3-[(3)H]pyruvate catalyzed by YPK occurs at a rate significantly greater than the spontaneous rate. Detritiation of pyruvate by wild-type YPK occurs as a divalent metal- and FBP-dependent process requiring ATP. There is no detectable detritiation of pyruvate catalyzed by K240M. The solvent deuterium isotope effect on k(cat) is 2.7 +/- 0.2 and 1.6 +/- 0.1 for the wild type and for K240M YPK, respectively. This suggests that the isotope sensitive step in the PK reaction does not involve Lys 240 and that the enolpyruvate intermediate is still protonated by K240M. Isotope trapping was used to characterize enolpyruvate protonation by K240M. While there was enrichment of the methyl protons of pyruvate from labeled solvent formed by catalysis with muscle PK and wild-type YPK, only background levels of tritium were trapped with K240M. In K240M, the proton donor exchanges protons with the solvent at a higher rate relative to turnover than does the proton donor in wild-type YPK. The pH-rate profile of K240M exhibits the loss of a pK(a) value of 8. 8 observed with wild-type YPK. The above data and recent crystal structure data suggest that Lys 240 interacts with the phosphoryl group of phosphoenolpyruvate and helps to stabilize the pentavalent phosphate transition state during phosphoryl transfer. Phosphoryl transfer is highly coupled to proton transfer, or Lys 240 also affects enolate protonation.
采用定点诱变技术将酵母丙酮酸激酶的赖氨酸240（肌肉丙酮酸激酶中的赖氨酸269）替换为甲硫氨酸。K240M突变体在催化过程中对果糖-1,6-二磷酸（FBP）有绝对需求。在分别存在锰离子（Mn(2+)）和镁离子（Mg(2+)）的情况下，K240M突变体的活性分别比野生型酵母丙酮酸激酶（YPK）低100倍和1000倍。稳态荧光滴定数据表明，底物磷酸烯醇式丙酮酸（PEP）与K240M突变体的结合亲和力与野生型YPK相同。与野生型YPK相比，K240M突变体中磷酸转移的速率降低了1000倍以上。YPK催化的3-[(3)H]丙酮酸的氚消除速率显著高于自发消除速率。野生型YPK催化的丙酮酸氚消除是一个依赖于二价金属离子和FBP且需要ATP的过程。K240M突变体催化的丙酮酸没有可检测到的氚消除现象。野生型和K240M突变体的溶剂氘同位素效应（对催化常数k(cat)）分别为2.7±0.2和1.6±0.1。这表明丙酮酸激酶反应中对同位素敏感的步骤不涉及赖氨酸240，并且烯醇丙酮酸中间体在K240M突变体中仍然被质子化。采用同位素捕获技术来表征K240M突变体对烯醇丙酮酸的质子化作用。在用肌肉丙酮酸激酶和野生型YPK催化形成的标记溶剂中，丙酮酸的甲基质子有富集现象，而用K240M突变体催化时，仅捕获到背景水平的氚。在K240M突变体中，质子供体与溶剂交换质子的速率相对于周转数而言比野生型YPK中的质子供体更高。K240M突变体的pH-速率曲线显示，失去了野生型YPK中观察到的pK(a)值8.8。上述数据和最近的晶体结构数据表明，赖氨酸240与磷酸烯醇式丙酮酸的磷酰基相互作用，并在磷酸转移过程中有助于稳定五价磷酸过渡态。磷酸转移与质子转移高度偶联，或者赖氨酸240也影响烯醇化物的质子化。
