Freist W, Sternbach H, Cramer F
Max-Planck-Institut für experimentelle Medizin, Göttingen, Germany.
Eur J Biochem. 1994 Mar 15;220(3):745-52. doi: 10.1111/j.1432-1033.1994.tb18675.x.
For discrimination between threonine and 18 other naturally occurring non-cognate amino acids by the class II aminoacyl-tRNA synthetase specific for threonine, discrimination factors (D) have been determined from Kca and Km values. The lowest values were found for Cys, Met, Val (D = 70-280), indicating that threonine is only 70-280-times more often esterified to tRNA(Thr)-C-C-A than are these non-cognate compounds at the same amino acid concentrations. The highest D values have been observed for Gly, Pro, Gln, Leu, Phe, and Lys (D = 1000-2000), for the other non-cognate amino acids D values are in the medium range 300-1000. Generally, threonyl-tRNA synthetase is less specific than the class I enzymes specific for Ile, Val, Tyr, Arg, but more specific than the only investigated class II enzyme specific for Lys. In aminoacylation of tRNA(Thr)-C-C-A(2'NH2) discrimination factors D1 are in the range 2-170. From D1 values and AMP-formation stoichiometry, pre-transfer proof-reading factors II1, were determined; post-transfer proof-reading factors II2 were determined from D values and AMP-formation stoichiometry in acylation of tRNA(Thr)-C-C-A. II1 values are in the range 1.8-33, II2 values in the range 1.4-22, thus threonyl-tRNA synthetase shows the highest post-transfer proof-reading activity of six investigated synthetases (specific for Ile, Val, Tyr, Arg, Lys). Initial discrimination factors caused by differences in Gibbs free energies of binding between threonine and non-cognate amino acids have been calculated from discrimination and proof-reading factors. Assuming a two-step binding process, two factors (I1 and I2) have been determined which can be related to hydrophobic interaction forces depending on accessible surface areas of the amino acids. The threonine side chain must be bound by hydrophobic forces and two hydrogen bonds. In contrast to proof-reading factors obtained with the synthetases specific for Ile, Val, Tyr, Arg, and Lys, proof-reading factors II1 and II2 obtained with threonyl-tRNA synthetase are also related to hydrophobic interaction of the amino acid side chains and the enzyme. Threonyl-tRNA synthetase examines side chain structures of amino acids in the four postulated recognition steps, for each step the enzyme uses special distinct structures or conformations of the binding cleft.
对于由苏氨酸特异性的II类氨酰 - tRNA合成酶区分苏氨酸与其他18种天然存在的非同源氨基酸，已根据Kca和Km值确定了区分因子（D）。半胱氨酸、甲硫氨酸、缬氨酸的区分因子最低（D = 70 - 280），这表明在相同氨基酸浓度下，苏氨酸与tRNA(Thr)-C-C-A酯化的频率仅比这些非同源化合物高70 - 280倍。甘氨酸、脯氨酸、谷氨酰胺、亮氨酸、苯丙氨酸和赖氨酸的D值最高（D = 1000 - 2000），其他非同源氨基酸的D值处于300 - 1000的中等范围。一般来说，苏氨酰 - tRNA合成酶的特异性低于异亮氨酸、缬氨酸、酪氨酸、精氨酸特异性的I类酶，但高于唯一研究的赖氨酸特异性的II类酶。在tRNA(Thr)-C-C-A(2'NH2)的氨酰化过程中，区分因子D1在2 - 170范围内。根据D1值和AMP形成化学计量比，确定了转移前校对因子II1；通过tRNA(Thr)-C-C-A酰化中的D值和AMP形成化学计量比确定了转移后校对因子II2。II1值在1.8 - 33范围内，II2值在1.4 - 22范围内，因此苏氨酰 - tRNA合成酶在六种研究的合成酶（异亮氨酸、缬氨酸、酪氨酸、精氨酸、赖氨酸特异性）中显示出最高的转移后校对活性。由苏氨酸与非同源氨基酸结合的吉布斯自由能差异引起的初始区分因子已根据区分和校对因子计算得出。假设为两步结合过程，确定了两个因子（I1和I2），它们可根据氨基酸的可及表面积与疏水相互作用力相关。苏氨酸侧链必须通过疏水力和两个氢键结合。与异亮氨酸、缬氨酸、酪氨酸、精氨酸和赖氨酸特异性的合成酶获得的校对因子不同，苏氨酰 - tRNA合成酶获得的校对因子II1和II2也与氨基酸侧链和酶的疏水相互作用有关。苏氨酰 - tRNA合成酶在四个假定的识别步骤中检查氨基酸的侧链结构，对于每个步骤，酶使用结合裂隙的特殊不同结构或构象。
