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首页> 外文期刊>Biochemistry >Induced Fit and Kinetic Mechanism of Adenylation Catalyzed by Escherichia coli Threonyl-tRNA Synthetase
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Induced Fit and Kinetic Mechanism of Adenylation Catalyzed by Escherichia coli Threonyl-tRNA Synthetase

机译：大肠杆菌苏糖酰基-tRNA合成酶催化腺苷酸化的诱导适应性和动力学机理

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Threonyl-tRNA synthetase (ThrRS) must discriminate among closely related amino acids to maintain the fidelity of protein synthesis.Here,a pre-steady state kinetic analysis of the ThRS-catalyzed adenylati8on reactio nwas carried out by monitoring changes in intrinsic tryptophan fluorescence.Stopped flow fluorimetry for the forward reaction gave a saturable fluorescence quench whose apparent rate increased hyperbolically with ATP cocnentration,consistent with a two-step mechanism in which rapid substrate binding precedes an isomerization step.From similar experiments,the equilibrium dissociation constants for dissociation of ATP from the E·Thr complex (K_3=450+-180 muM) and threonine from the E·ATP complex (K'_4=135 muM) and the forward rate cosntant for adenylation (k+(+5)=29+-4 s~(-1)) were determined.A saturable fluorescence increase accompanied the pyrophosphorolysis of the E·Thr approx AMP complex,affording the dissociation constant for PP_i(K_6=170+-50muM) and the reverse rate constant (k_(-5)=47+-4 s~(-1)).The longer side chain of beta-hydroxynorvaline increased the apparent dissociation constant (K_(4[HNV]=6.8+-2.8 mM) with only a small reduction i nthe forward rate (k'-(5[HIV])=20+-3.1 s~(-1)).In contrast,two nonproductive substrates,threoninol and the adenylate analogue 5'-O-[N-(L-threonyl)sulfamoyl]-adenosine (Thr-AMS),exhibited linear increases in k_(app) with lgiand concentration,suggesting that their binding is slow relative to isomerization.The proposed mechanism is consistent with steady state kinetic parameters.The role of threonine binding loop residue Trp434 in fluorescence changes was established by mutagenesis.The combined kinetic and molecular genetic analyses presented here support the principle of induced fit in the ThrRS-catalyzed adenylation reaction,in which substrate binding drives conformational changes that orient substrates and active site groups for catalysis.
机译：苏糖基-tRNA合成酶（ThrRS）必须区分密切相关的氨基酸以维持蛋白质合成的保真度。在此，通过监测固有色氨酸荧光的变化，对ThRS催化的腺苷酸反应进行了稳态前动力学分析。流式荧光法检测正向反应的饱和荧光猝灭，其表观速率随ATP浓度双曲线增加，这与两步机理有关，在该机理中，快速的底物键合先于异构化步骤。根据类似的实验，ATP从中解离的平衡解离常数E·Thr络合物（K_3 = 450 + -180μM）和苏氨酸从E·ATP络合物（K'_4 = 135μM）和腺苷酸的顺向速率余量（k +（+ 5）= 29 + -4 s〜确定（-1））。伴随着E·Thr约AMP配合物的热磷解，饱和荧光增加，满足PP_i（K_6 = 170 +-50μM）的解离常数和反向速率常数。 tant（k _（-5）= 47 + -4 s〜（-1））。更长的β-羟基降冰片侧链增加了表观解离常数（K_（4 [HNV] = 6.8 + -2.8 mM）前进速率的小降低（k'-（5 [HIV]）= 20 + -3.1 s〜（-1））。相比之下，两种非生产性底物苏氨酸和腺苷酸类似物5'-O- [N-（ L-苏氨酰氨磺酰基]-腺苷（Thr-AMS）在k_（app）随lgi和浓度的增加呈线性增加，这表明它们的结合相对于异构化是缓慢的。提出的机理与稳态动力学参数是一致的。通过诱变建立了苏氨酸结合环残基Trp434的荧光变化。这里提出的动力学和分子遗传学联合分析支持了ThrRS催化的腺苷酸化反应的诱导契合原理，其中底物结合驱动构象变化，从而定向底物和活性位点组用于催化。

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