Zhi W, Srere P A, Evans C T
Pre-Clinical Science Unit, Department of Veterans Affairs Medical Center, Dallas, Texas.
Biochemistry. 1991 Sep 24;30(38):9281-6. doi: 10.1021/bi00102a021.
The conformational stabilities of native pig citrate synthase (PCS), a recombinant wild-type PCS, and six active-site mutant pig citrate synthases were studied in thermal denaturation experiments by circular dichroism and in urea denaturation experiments by using DTNB to measure the appearance of latent SH groups. His274 and Asp375 are conserved active-site residues in pig citrate synthase that bind to substrates and are implicated in the catalytic mechanism of the enzyme. By site-directed mutagenesis, His274 was replaced with Gly and Arg, while Asp375 was replaced with Gly, Asn, Glu, or Gln. These modifications were previously shown to result in 10(3)-10(4)-fold reductions in enzyme specific activities. The thermal unfolding of pig citrate synthase and the six mutants in the presence and absence of substrates showed large differences in the thermal stabilities of mutant proteins compared to the wild-type pig citrate synthase. The functions of His274 and Asp375 in ligand binding were measured by oxalacetate protection against urea denaturation. These data indicate that active-site mutations that decrease the specific activity of pig citrate synthase also cause an increase in the conformational stability of the protein. These results suggest that specific electrostatic interactions in the active site of citrate synthase are important in the catalytic mechanism in the chemical transformations as well as the conformational flexibility of the protein, both of which are important for the overall catalytic efficiency of the enzyme.
通过圆二色性进行热变性实验，以及使用5,5'-二硫代双(2-硝基苯甲酸)(DTNB)测量潜在巯基的出现来进行尿素变性实验，研究了天然猪柠檬酸合酶(PCS)、重组野生型PCS和六种活性位点突变猪柠檬酸合酶的构象稳定性。His274和Asp375是猪柠檬酸合酶中保守的活性位点残基，它们与底物结合并参与酶的催化机制。通过定点诱变，将His274替换为甘氨酸和精氨酸，同时将Asp375替换为甘氨酸、天冬酰胺、谷氨酸或谷氨酰胺。这些修饰先前已显示会导致酶比活性降低10³-10⁴倍。在有和没有底物存在的情况下，猪柠檬酸合酶和六个突变体的热解折叠显示，与野生型猪柠檬酸合酶相比，突变蛋白的热稳定性存在很大差异。通过草酰乙酸对尿素变性的保护作用来测量His274和Asp375在配体结合中的功能。这些数据表明，降低猪柠檬酸合酶比活性的活性位点突变也会导致蛋白质构象稳定性增加。这些结果表明，柠檬酸合酶活性位点中的特定静电相互作用在化学转化的催化机制以及蛋白质的构象灵活性中都很重要，这两者对酶的整体催化效率都很重要。
