Freist W, Sternbach H, Cramer F
Max-Planck-Institut für experimentelle Medizin, Göttingen.
Eur J Biochem. 1992 Mar 15;204(3):1015-23. doi: 10.1111/j.1432-1033.1992.tb16723.x.
Discrimination factors (D) which are characteristic for discrimination between lysine and 19 naturally occurring non-cognate amino acids have been determined from kcat and Km values for native and phosphorylated lysyl-tRNA synthetases from yeast. Generally, both species of this class II aminoacyl-tRNA synthetase are considerably less specific than the class I synthetases specific for isoleucine, valine, tyrosine, and arginine. D values of the native enzyme are in the range 90-1700, D values of the phosphorylated species in the range 40-770. The phosphorylated enzyme acts faster and less accurately. In aminoacylation of tRNALys-C-C-A(2'NH2) discrimination factors D1 vary over 30-980 for the native and over 8-300 for the phosphorylated enzyme. From AMP formation stoichiometry and D1 values pretransfer proof-reading factors (II1) of 1.1-56 were calculated for for the native enzyme, factors of 1.0-44 for the phosphorylated species. Post-transfer proof-reading factors (II2) were calculated from D values and AMP formation stoichiometry in acylation of tRNALys-C-C-A. Pretransfer proof-reading is the main correction step, posttransfer proof-reading is less effective or negligible (II2 approximately 1-8). Initial discrimination factors (I), which are due to differences in Gibbs free energies of binding between lysine and noncognate substrates (delta delta GI), were calculated from discrimination and proof-reading factors. In contrast to class I synthetases, for lysyl-tRNA synthetase only one initial discrimination step can be assumed and amino acid recognition is reduced to a three-step process instead of the four-step recognition observed for the class I synthetases. Plots of delta delta GI values against accessible surface areas of amino acids show clearly that phosphorylation of the enzyme changes the structures of the amino acid binding sites. This is illustrated by a hypothetical 'stopper model' of these sites.
已根据酵母中天然和磷酸化赖氨酰 - tRNA合成酶的kcat和Km值，确定了用于区分赖氨酸与19种天然存在的非同源氨基酸的区分因子（D）。一般来说，这类II类氨酰 - tRNA合成酶的两种形式都比I类对异亮氨酸、缬氨酸、酪氨酸和精氨酸具有特异性的合成酶特异性低得多。天然酶的D值在90 - 1700范围内，磷酸化形式的D值在40 - 770范围内。磷酸化酶作用更快但准确性更低。在tRNALys - C - C - A(2'NH2)的氨酰化过程中，天然酶的区分因子D1在30 - 980范围内变化，磷酸化酶的D1在8 - 300范围内变化。根据AMP形成化学计量和D1值，计算出天然酶的转移前校对因子（II1）为1.1 - 56，磷酸化形式的为1.0 - 44。转移后校对因子（II2）是根据tRNALys - C - C - A酰化过程中的D值和AMP形成化学计量计算得出的。转移前校对是主要的校正步骤，转移后校对效果较差或可忽略不计（II2约为1 - 8）。初始区分因子（I）是由于赖氨酸与非同源底物之间结合吉布斯自由能的差异（δΔGI），由区分和校对因子计算得出。与I类合成酶不同，对于赖氨酰 - tRNA合成酶，只能假设一个初始区分步骤，氨基酸识别简化为三步过程，而不是I类合成酶中观察到的四步识别过程。δΔGI值相对于氨基酸可及表面积的图清楚地表明，酶的磷酸化改变了氨基酸结合位点的结构。这些位点的一个假设“塞子模型”对此进行了说明。
