KINETICS AND THERMODYNAMICS OF THE INTERACTION OF 5-FLUORO-2'-DEOXYURIDYLATE WITH THYMIDYLATE SYNTHASE
KINETICS AND THERMODYNAMICS OF THE INTERACTION OF 5-FLUORO-2'-DEOXYURIDYLATE WITH THYMIDYLATE SYNTHASE
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DOI：
10.1021/bi00400a017
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发表时间：
1987-12-29
期刊：
影响因子：
2.9
通讯作者：
IVANETICH, KM
中科院分区：
文献类型：
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作者：
SANTI, DV;MCHENRY, CS;IVANETICH, KM
胸苷合成酶(TS)、5-氟代脱氧尿酸(FdUMP)和5，10-亚甲基四氢叶酸(CH2-H4)形成共价复合体，其中TS的半胱硫醇连接到FdUMP的6位，辅因子的1碳单元连接到5位。用半快冷法测定了该共价络合物在不同温度下的生成动力学。结合以前报道的数据，这些结果可以计算相互作用中的每个速率和平衡常数。在25℃时，非共价三元络合物转化为相应共价络合物的速率为0.6%S~(-1)。C，从非共价三元络合物中损失CH2-H4叶酸的离解常数为.apprx。形成共价络合物的活化参数为：E_a=20千卡/摩尔，ΔG+=17.9千卡/摩尔，ΔH·dbldag。=19.3千卡/摩尔，ΔS·dbldag。=0.005千卡/(摩尔度)。非共价和共价三元络合物之间的平衡常数为.apprx。2.泰晤士报。104，CH2-H4叶酸与共价络合物的总解离常数为.apprx。10-11M，在正常的酶反应中，非共价三元络合物转化为共价加合物的速度比kcat慢约12倍。然而，由于CH2-H4叶酸从非共价三元络合物中的离解常数比TS-Dump-CH2-H4叶酸胶束络合物中的离解常数低约10倍，所以与kcat/Km相对应的项几乎相等。我们认为，CH2-H4叶酸的一些固有结合能可能被用来促进5-亚胺离子中间体的形成。我们认为亚胺离子的形成是通过(A)CH2-H4叶酸在N-10位的一般酸催化和(B)酶诱导的非共价TS-FdUMP-CH2-H4叶酸络合物中辅因子五元环的扰动来催化的。后者可能涉及酶的总酸催化剂与N-10的氢键，辅因子的对氨基苯甲酸部分的扰动，以及辅因子的五元环上的张力。
Thymidylate synthase (TS), 5-fluorodeoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (CH2-H4folate) form a covalent complex in which a Cys thiol of TS is attached to the 6-position of FdUMP and the one-carbon unit of the cofactor is attached to the 5-position. The kinetics of formation of this covalent complex have been determined at several temperatures by semirapid quench methods. Together with previously reported data the results permit calculation of every rate and equilibrium constant in the interaction. Conversion of the noncovalent ternary complex to the corresponding covalent complex proceeds at a rate of 0.6 s-1 at 25.degree. C, and the dissociation constant for loss of CH2-H4 folate from the noncovalent ternary complex is .apprx. 1 .mu.M. Activation parameters for the formation of the covalent complex were shown to be Ea = 20 kcal/mol, .DELTA.G+ = 17.9 kcal/mol, .DELTA.H.dbldag. = 19.3 kcal/mol, and .DELTA.S.dbldag. = 0.005 kcal/(mol-deg). The equilibrium constant between the noncovalent and covalent ternary complexes is .apprx. 2 .times. 104, and the overall dissociation constant of CH2-H4folate from the covalent complex is .apprx. 10-11 M. The conversion of the noncovalent ternary complex to the covalent adduct is about 12-fold slower than kcat in the normal enzymic reaction. However, because the dissociation constant for CH2-H4folate from the noncovalent ternary complex is about 10-fold lower than that from the TS-dUMP-CH2-H4folate Michaelis complex, the terms corresponding to kcat/Km are nearly equal. We propose that some of the intrinsic binding energy of CH2-H4folate may be used to facilitate formation of a 5-iminium ion intermediate. We suggest that iminium ion formation is catalyzed by (a) general-acid catalysis at N-10 of CH2-H4folate and (b) enzyme-induced perturbations of the five-membered ring of the cofactor within the noncovalent TS-FdUMP-CH2-H4folate complex. The latter may involve hydrogen bonding of the enzyme general-acid catalyst to N-10, perturbation of the p-aminobenzoic acid moiety of the cofactor, and strain on the five-membered ring of the cofactor.