Kinetic scheme for thymidylate synthase from Escherichia coli: Determination from measurements of ligand binding, primary and secondary isotope effects, and pre-steady-state catalysis
Kinetic scheme for thymidylate synthase from Escherichia coli: Determination from measurements of ligand binding, primary and secondary isotope effects, and pre-steady-state catalysis
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DOI：
10.1021/bi961794q
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发表时间：
1997-04-08
期刊：
影响因子：
2.9
通讯作者：
Appleman, JR
中科院分区：
文献类型：
--
作者：
Spencer, HT;Villafranca, JE;Appleman, JR
我们已经确定了动力学和热力学常数，足以描述这种酶的动力学方案的底物和产品的结合胸苷酸合酶从大肠杆菌（TS）。(1)催化机理顺序为：TS + dUMP → TS .dUMP +（6 R）-5，10-CH_2-H（4）folate → TS. dUMP。（GR）-5，10-CH（2）H（4）folate --> TS.dTMP.H（2）folate --> TS .dTMP --> TS，如其他人先前从稳态测量中预测的。（2）当底物饱和时，总反应速率受酶结合底物向结合产物的缓慢转化控制，如（i）对k（cat）的大的初级和次级同位素效应和（ii）与k（cat）相比的高产物解离速率所证明的。(3)停流研究测量了10-炔丙基-5，8-二去氮唑酸盐（6 R）-5，10-CH（2）H（4）叶酸的类似物）与活性位点突变体C146 A或C-末端截短突变体P261 Am的结合，使我们能够鉴定与野生型酶在催化起始期间观察到的光谱变化相对应的物理事件。一个动力学可识别的反应步骤，TS. dUMP。（6R）-5，10-CH（2）H（4）folate -->（TS.dUMP.（6 R）-5，10-CH（2）H（4）叶酸）*，可能代表酶的C-末端在催化位点上的重定向。这将基材密封在相对非水性的环境中，在该环境中可以发生催化。(4)虽然TS是相同亚基的二聚体，但催化可能一次仅限于一个亚基。（5）本文所述的“高分辨率”动力学方案提供了一个框架，用于解释突变体ecTS的催化动力学，所述突变体ecTS被选择以提供对结构和功能之间关系的见解。
We have determined kinetic and thermodynamic constants governing binding of substrates and products to thymidylate synthase from Escherichia coli (TS) sufficient to describe the kinetic scheme for this enzyme. (1) The catalytic mechanism is ordered in the following manner, TS + dUMP --> TS .dUMP + (6R)-5, 10-CH2-H(4)folate --> TS.dUMP.(GR)-5,10-CH(2)H(4)folate --> TS.dTMP.H(2)folate --> TS .dTMP --> TS as predicted previously by others from steady-state measurements. (2)When substrates are saturating, the overall reaction rate is governed by the slow conversion of enzyme-bound substrates to bound products as demonstrated by (i) large primary and secondary isotope effects on k(cat) and (ii) high rates of product dissociation compared to k(cat). (3) Stopped-flow studies measuring the binding of 10-propargyl-5,8-dideazafolate, an analog of (6R)-5,10-CH(2)H(4)folate, with the active site mutant C146A or the C-terminus-truncated mutant P261Am enabled us to identify physical events corresponding to spectral changes which are observed with the wild-type enzyme during initiation of catalysis. A kinetically identifiable reaction step, TS.dUMP.(6R)-5,10-CH(2)H(4)folate --> (TS.dUMP.(6R)-5,10-CH(2)H(4)folate)*, likely represents reorientation of the C-terminus of the enzyme over the catalytic site. This seals the substrates into a relatively nonaqueous environment in which catalysis can occur. (4) Although TS is a dimer of identical subunits, catalysis is probably confined to only one subunit at a time, (5) The ''high-resolution'' kinetic scheme described herein provides a framework for the interpretation of the kinetics of catalysis by mutant ecTS chosen to provide insights into the relationship between structure and function.