Likos J J, Hess B, Colman R F
J Biol Chem. 1980 Oct 10;255(19):9388-98.
Yeast pyruvate kinase is irreversibly inactivated by 1.1 mM 5'-p-fluorosulfonylbenzoyl adenosine at pH 8.6 with an initial rate constant of 0.019 min-1. A plot of kinact versus the 5'-p-fluorosulfonylbenzoyl adenosine concentration yields a hyperbolic curve indicative of binding of the analog prior to reaction. Marked protection is afforded by phosphoenolpyruvate + fructose 1,6-diphosphate + Mg2+ or MgATP suggesting that reaction occurs within the active site. When assayed at less than saturating phosphoenolpyruvate concentrations, the inactivation caused by the reagent in the absence of added ligands appears slower, and reaction in the presence of phosphoenolpyruvate, fructose 1,6-diphosphate, and Mg2+ produces an activation of the enzyme, the extent of which is dependent on the assay concentration of phosphoenolpyruvate. The rate constant for activation was observed to be 0.113 min-1. The activated enzyme exhibits both a lowered K0.5 and Hill coefficient compared to native pyruvate kinase. Subsequent addition of 5'-p-fluorosulfonylbenzoyl adenosine to activated pyruvate kinase in the absence of added ligands leads to inactivation with the rate constant independent of the assay concentration of phosphoenolpyruvate. Covalent reaction of pyruvate kinase with 5'-p-fluorosulfonylbenzoyl adenosine thus occurs at two distinct sites. In the presence of phosphoenolpyruvate, fructose 1,6-diphosphate, and Mg2+, incorporation of tritiated 5'-p-fluorosulfonylbenzoyl adenosine is linearly proportional to the extent of activation of the enzyme, with 4 mol of reagent bound/mol of tetrameric pyruvate kinase for maximally activated enzyme. In the absence of added ligands, approximately 4.5 mol of reagent are incorporated/mol of enzyme at 15 min of reaction, while 80% of the original activity remains. Subsequent incorporation is proportional to the extent of inactivation with 8 mol bound at 100% in activaton. In the presence of phosphoenolpyruvate, fructose 1,6-diphospate, and Mg2+, 3 tyrosines and 1 lysine residue, and in the absence of ligands, 6 tyrosines and 2 lysine residues are modified, suggesting that both amino acids are within the two nucleotide sites.
在pH 8.6条件下，1.1 mM的5'-对氟磺酰苯甲酰腺苷可使酵母丙酮酸激酶不可逆地失活，初始速率常数为0.019 min⁻¹。以酶促反应最大速率（kinact）对5'-对氟磺酰苯甲酰腺苷浓度作图，得到一条双曲线，表明该类似物在反应前发生了结合。磷酸烯醇丙酮酸+果糖1,6-二磷酸+Mg²⁺或MgATP可提供显著的保护作用，这表明反应发生在活性位点内。当在低于饱和的磷酸烯醇丙酮酸浓度下进行测定时，在没有添加配体的情况下，该试剂引起的失活似乎较慢，而在磷酸烯醇丙酮酸、果糖1,6-二磷酸和Mg²⁺存在的情况下反应会使酶激活，激活程度取决于磷酸烯醇丙酮酸的测定浓度。观察到激活的速率常数为0.113 min⁻¹。与天然丙酮酸激酶相比，激活后的酶表现出较低的半最大激活浓度（K0.5）和希尔系数。在没有添加配体的情况下，随后向激活的丙酮酸激酶中加入5'-对氟磺酰苯甲酰腺苷会导致失活，失活速率常数与磷酸烯醇丙酮酸的测定浓度无关。因此，丙酮酸激酶与5'-对氟磺酰苯甲酰腺苷的共价反应发生在两个不同的位点。在磷酸烯醇丙酮酸、果糖1,6-二磷酸和Mg²⁺存在的情况下，氚标记的5'-对氟磺酰苯甲酰腺苷的掺入量与酶的激活程度呈线性比例关系，对于最大激活的酶，每摩尔四聚体丙酮酸激酶结合4摩尔试剂。在没有添加配体的情况下，反应15分钟时，每摩尔酶大约掺入4.5摩尔试剂，而仍保留80%的原始活性。随后的掺入量与失活程度成比例，在100%失活时结合8摩尔。在磷酸烯醇丙酮酸、果糖1,6-二磷酸和Mg²⁺存在的情况下，3个酪氨酸和1个赖氨酸残基被修饰，在没有配体的情况下，6个酪氨酸和2个赖氨酸残基被修饰，这表明这两种氨基酸都在两个核苷酸位点内。
