Tomich J M, Marti C, Colman R F
Biochemistry. 1981 Nov 10;20(23):6711-20. doi: 10.1021/bi00526a029.
Reaction of rabbit muscle pyruvate kinase with the affinity label 5'-[p-(fluorosulfonyl) benzoyl] guanosine (5'-FSBG), at pH 7.65 and 7.93, leads to a loss in enzyme activity. The inactivation is characterized by a biphasic kinetic profile, with the initial phase accounting for approximately 55% of the reduction in enzymatic activity. For both the rapid and slow phases, at pH 7.93, the inactivation rate constants are linearly proportional to the reagent concentration (from 0.48 to 3.0 mM), yielding second-order rate constants of 195 min-1 M-1 and 19 min-1 m-1, respectively. The effect of ligands was tested on the two phases of inactivation. For both, a decrease in the inactivation rate was produced by Mg2+ alone, but the best protection was provided by Mg2+ plus either ADP or GDP, suggesting that the reaction occurs in the region of the metal-nucleotide binding site. Modified pyruvate kinase is completely reactivated by incubation with 20 mM dithiothreitol, indicating the involvement of cysteine in the inactivation, indicating the involvement of cysteine in the inactivation process. Reaction with [5'=3H]-5'-FSBG leads to the incorporation of up to 1.3 mol of radioactive reagent per mol of enzyme subunit; however, identical radiolabel incorporation is observed before or after dithiothreitol reactivation of modified enzyme. This result implies that the labeled amino acid residue, measured by means of incorporation, is not directly involved in the inactivation process. In contrast, inactivation was found to correlate well with the loss of two free sulfhydryl groups per enzyme subunit and the restoration of activity to correlate with the regeneration of two free sulfhydryls after treatment of modified enzyme with dithiothreitol. It is proposed that inactivation of pyruvate kinase by 5'-FSBG proceeds by formation of thiol sulfonate followed by a rapid displacement of the sulfinic acid moiety by a second cysteine to yield a disulfide. A negative cooperatively in the interaction of pyruvate kinase subunits with 5'-[p-(fluorosulfonyl)-benzoyl] guanosine might best account for the biphasic inactivation kinetics.
在pH 7.65和7.93条件下，兔肌肉丙酮酸激酶与亲和标记物5'-[对-(氟磺酰基)苯甲酰基]鸟苷(5'-FSBG)反应会导致酶活性丧失。失活表现为双相动力学曲线，初始阶段约占酶活性降低的55%。在pH 7.93时，对于快速和慢速阶段，失活速率常数均与试剂浓度(0.48至3.0 mM)呈线性比例关系，二阶速率常数分别为195 min⁻¹ M⁻¹和19 min⁻¹ M⁻¹。测试了配体对失活两个阶段的影响。对于两者，单独的Mg²⁺会使失活速率降低，但Mg²⁺与ADP或GDP共同作用提供了最佳保护，这表明反应发生在金属-核苷酸结合位点区域。用20 mM二硫苏糖醇孵育可使修饰的丙酮酸激酶完全重新激活，表明半胱氨酸参与了失活过程。与[5'-³H]-5'-FSBG反应导致每摩尔酶亚基最多掺入1.3摩尔放射性试剂；然而，在修饰酶经二硫苏糖醇重新激活之前或之后观察到相同的放射性标记掺入。该结果表明，通过掺入测量的标记氨基酸残基不直接参与失活过程。相反，发现失活与每个酶亚基两个游离巯基的丧失密切相关，并且活性的恢复与用二硫苏糖醇处理修饰酶后两个游离巯基的再生相关。有人提出，5'-FSBG使丙酮酸激酶失活是通过形成硫醇磺酸酯，随后第二个半胱氨酸快速取代亚磺酸部分以产生二硫键。丙酮酸激酶亚基与5'-[对-(氟磺酰基)-苯甲酰基]鸟苷相互作用中的负协同作用可能最能解释双相失活动力学。
