Energetics of S-adenosylmethionine synthetase catalysis
Energetics of S-adenosylmethionine synthetase catalysis
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DOI：
10.1021/bi992876s
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发表时间：
2000-04-18
期刊：
影响因子：
2.9
通讯作者：
Markham, GD
中科院分区：
文献类型：
--
作者：
McQueney, MS;Anderson, KS;Markham, GD
s -腺苷蛋氨酸合成酶（ATP: l -蛋氨酸s -腺苷转移酶）催化了一级生物烷基化剂的唯一已知生物合成途径。大肠杆菌s -腺苷蛋氨酸（AdoMet）合成酶催化ATP、蛋氨酸和水形成AdoMet、焦磷酸盐（PPi）和磷酸盐（P-i）的内部热力学通过预稳态动力学、溶剂同位素掺入和平衡结合测量结合计算机建模来确定。这些研究提供了底物结合、两个化学相互转化步骤[AdoMet形成和随后的三聚磷酸盐（PPPi）水解]和产物释放的速率常数。数据表明，存在一个动力学显著的异构化e.a dome . ppi。产品发布前的P-i复合体。利用这些实验值和底物和产物的体内浓度，构建了生理条件下酶催化反应的自由能谱。自由能谱显示AdoMet生成反应的过渡态和基态能量不平衡，其平衡常数为10(4)。相反，随后的PPPi水解反应在能量上更好地平衡。热力学谱图表明，AdoMet的形成需要结合能的催化，并且PPPi的后续水解必须允许酶的周转。晶体学研究表明，一个可移动的蛋白质环可以进入活性位点。目前的动力学研究表明，相对于k（cat）和生理浓度下的底物结合，这种环运动是快速的。对于不同结构的配体，其一致的缓慢结合速率为10(4)-10(5)M-1 s(-1)，这表明环运动可能是蛋白质的固有特性，而不是配体诱导的。
S-Adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase) catalyzes the only known route of biosynthesis of the primary biological alkylating agent. The internal thermodynamics of the Escherichia coli S-adenosylmethionine (AdoMet) synthetase catalyzed formation of AdoMet, pyrophosphate (PPi), and phosphate (P-i) from ATP, methionine, and water have been determined by a combination of pre-steady-state kinetics, solvent isotope incorporation, and equilibrium binding measurements in conjunction with computer modeling. These studies provided the rate constants for substrate binding, the two chemical interconversion steps [AdoMet formation and subsequent tripolyphosphate (PPPi) hydrolysis], and product release. The data demonstrate the presence of a kinetically significant isomerization of the E.AdoMet.PPi.P-i complex before product release. The free energy profile for the enzyme-catalyzed reaction under physiological conditions has been constructed using these experimental values and in vivo concentrations of substrates and products. The free energy profile reveals that the AdoMet formation reaction, which has an equilibrium constant of 10(4), does not have well-balanced transition state and ground state energies. In contrast, the subsequent PPPi hydrolytic reaction is energetically better balanced, The thermodynamic profile indicates the use of binding energies for catalysis of AdoMet formation and the necessity for subsequent PPPi hydrolysis to allow enzyme turnover. Crystallographic studies have shown that a mobile protein loop gates access to the active site. The present kinetic studies indicate that this loop movement is rapid with respect to k(cat) and with respect to substrate binding at physiological concentrations, The uniformly slow binding rates of 10(4)-10(5) M-1 s(-1) for ligands with different structures suggest that loop movement may be an intrinsic property of the protein rather than being ligand induced.