Lodi P J, Chang L C, Knowles J R, Komives E A
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
Biochemistry. 1994 Mar 15;33(10):2809-14. doi: 10.1021/bi00176a009.
The role of lysine-12 at the active site of yeast triosephosphate isomerase has been elucidated by a combination of site-directed mutagenesis, Fourier transform infrared spectroscopy, enzyme kinetics, and X-ray crystallography. Several lines of evidence suggest that the mutant isomerase in which lysine has been changed to methionine cannot bind substrate. This mutant enzyme has no detectable catalytic activity, and infrared experiments show no evidence of binding dihydroxyacetone phosphate nor dihydroxyacetone sulfate to the active site. Furthermore, crystals of the enzyme grown in the presence of phosphoglycolohydroxamate, a potent reaction intermediate analog, show an open active site with no inhibitor bound. Mutation of lysine-12 to arginine produces a protein with a value Km elevated by a factor of 22, a Vmax reduced by a factor of 180, and a Ki for phosphoglycolohydroxamate elevated by a factor of 290. Mutation of lysine-12 to histidine produces an enzyme that shows virtually no catalytic activity at neutral pH, but below pH 6.1 this enzyme is active, suggesting that protonation of the histidine in this mutant is required for activity. These studies, together with the structural results reported in an accompanying paper, provide convincing evidence that a positive charge is required for substrate binding at the active site of triosephosphate isomerase and that lysine-12 provides this positive charge.
通过定点诱变、傅里叶变换红外光谱、酶动力学和X射线晶体学等方法相结合，已阐明了赖氨酸 - 12在酵母磷酸丙糖异构酶活性位点的作用。多条证据表明，赖氨酸已被甲硫氨酸取代的突变异构酶无法结合底物。这种突变酶没有可检测到的催化活性，红外实验表明没有证据显示磷酸二羟丙酮或硫酸二羟丙酮与活性位点结合。此外，在磷酸糖异羟肟酸（一种有效的反应中间类似物）存在下生长的该酶晶体显示出一个没有抑制剂结合的开放活性位点。将赖氨酸 - 12突变为精氨酸会产生一种蛋白质，其Km值升高了22倍，Vmax降低了180倍，磷酸糖异羟肟酸的Ki升高了290倍。将赖氨酸 - 12突变为组氨酸会产生一种在中性pH下几乎没有催化活性的酶，但在pH 6.1以下该酶具有活性，这表明该突变体中组氨酸的质子化是活性所必需的。这些研究，连同随附论文中报道的结构结果，提供了令人信服的证据，即磷酸丙糖异构酶活性位点的底物结合需要正电荷，而赖氨酸 - 12提供了这种正电荷。
