Hu C Q, Sturtevant J M
Department of Chemistry, Yale University, New Haven, Connecticut 06511.
Biochemistry. 1989 Jan 24;28(2):813-8. doi: 10.1021/bi00428a060.
In continuation of earlier work [Hu, C. Q., & Sturtevant, J.M. (1987) Biochemistry 26, 178-182], differential scanning calorimetry has been employed in a study of the effects on the thermal denaturation of yeast phosphoglycerate kinase of two inhibitors of the enzyme, sulfate ion and the dye Cibracron blue F3GA. Sulfate ion, as is usual with ligands that dissociate during unfolding of the host protein, raises t1/2, the temperature of half-completion of the denaturation, has only a modest effect, stemming from the enthalpy of dissociation of the ligand, on the enthalpy of denaturation, and has little or no effect on the heat capacity change resulting from denaturation. In sharp contrast, Cibacron blue F3GA lowers t1/2 and drastically decreases both the enthalpy and heat capacity changes due to denaturation. The DSC results with sulfate ion are consistent with previous kinetic data [Scopes, R. K. (1978) Eur. J. Biochem. 91, 119-129; Khamis, M. H., & Larsson-Raznikiewicz, M. (1981) Biochim. Biophys. Acta 657, 190-194], which indicate two binding sites for sulfate ion at one of which the ligand acts as a competitive inhibitor. The results with Cibacron blue F3GA indicate that the dye induces a major destabilizing structural change in the enzyme in addition to rendering it enzymically inactive.
作为早期工作[Hu, C. Q., & Sturtevant, J.M. (1987) Biochemistry 26, 178 - 182]的延续，差示扫描量热法被用于研究该酶的两种抑制剂——硫酸根离子和染料Cibracron blue F3GA对酵母磷酸甘油酸激酶热变性的影响。硫酸根离子，与在宿主蛋白展开过程中解离的配体情况一样，提高了t1/2（变性完成一半时的温度），由于配体解离焓的作用，对变性焓只有适度影响，并且对变性导致的热容变化几乎没有影响。与之形成鲜明对比的是，Cibacron blue F3GA降低了t1/2，并显著降低了由于变性导致的焓变和热容变化。硫酸根离子的差示扫描量热结果与先前的动力学数据[Scopes, R. K. (1978) Eur. J. Biochem. 91, 119 - 129; Khamis, M. H., & Larsson - Raznikiewicz, M. (1981) Biochim. Biophys. Acta 657, 190 - 194]一致，这些数据表明硫酸根离子有两个结合位点，其中一个位点上配体作为竞争性抑制剂起作用。Cibacron blue F3GA的结果表明，除了使酶失去酶活性外，该染料还在酶中诱导了主要的不稳定结构变化。
