Wells T N, Knill-Jones J W, Gray T E, Fersht A R
MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, U.K.
Biochemistry. 1991 May 28;30(21):5151-6. doi: 10.1021/bi00235a006.
The first step of the reaction catalyzed by the aminoacyl-tRNA synthetases is the formation of enzyme-bound aminoacyl adenylate. The steady-state kinetics of this step has conventionally been studied by measuring the rate of isotopic exchange between pyrophosphate and ATP. A simple kinetic analysis of the pyrophosphate-exchange reaction catalyzed by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus is given in which all the observed rate and binding constants can be assigned to identifiable physical processes under a variety of limiting conditions. The free energies of binding to the enzyme of tyrosine, ATP, and the transition state for tyrosyl adenylate formation can be measured in relatively straightforward experiments. The excellent agreement between parameters measured in these experiments and those from earlier pre-steady-state kinetics confirms that the intermediates isolated in the presteady state are kinetically competent. The dissociation constant of ATP from the unligated enzyme, a constant that has previously been experimentally inaccessible, has been measured for wild-type and several mutant enzymes. The changes in enthalpy and entropy of activation on mutation have been measured by a rapid procedure for mutants that have altered contacts with tyrosine and ATP. Those mutants that have large changes of enthalpy and entropy of binding are likely to have structural changes and so warrant further examination by protein crystallography.
氨酰 - tRNA合成酶催化的反应的第一步是形成酶结合的氨酰腺苷酸。这一步的稳态动力学传统上是通过测量焦磷酸和ATP之间的同位素交换速率来研究的。本文给出了嗜热脂肪芽孢杆菌酪氨酰 - tRNA合成酶催化的焦磷酸交换反应的简单动力学分析，其中所有观察到的速率和结合常数都可以在各种极限条件下归因于可识别的物理过程。酪氨酸、ATP与酶的结合自由能以及酪氨酰腺苷酸形成的过渡态自由能可以通过相对简单的实验来测量。这些实验中测量的参数与早期预稳态动力学的参数之间的良好一致性证实了在预稳态中分离出的中间体在动力学上是有活性的。已经测量了野生型和几种突变酶中ATP从未结合酶上的解离常数，这是一个以前无法通过实验获得的常数。对于与酪氨酸和ATP接触发生改变的突变体，通过一种快速方法测量了突变时活化焓和活化熵的变化。那些结合焓和熵有很大变化的突变体可能有结构变化，因此值得通过蛋白质晶体学进一步研究。
