1EV8

CRYSTAL STRUCTURE ANALYSIS OF CYS167 MUTANT OF ESCHERICHIA COLI

1EV8 の概要

• エントリーDOI: 10.2210/pdb1ev8/pdb
• 関連するPDBエントリー: 1EV5 1EVF 1EVG
• 分子名称: THYMIDYLATE SYNTHASE (2 entities in total)
• 機能のキーワード: cys167 e. coli thymidylate synthase, transferase
• 由来する生物種: Escherichia coli
• 細胞内の位置: Cytoplasm : P0A884
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 30880.20

構造登録者

Phan, J.,Mahdavian, E.,Nivens, M.C.,Minor, W.,Berger, S.,Spencer, H.T.,Dunlap, R.B.,Lebioda, L. (登録日: 2000-04-19, 公開日: 2000-05-03, 最終更新日: 2022-04-13)

主引用文献

Phan, J.,Mahdavian, E.,Nivens, M.C.,Minor, W.,Berger, S.,Spencer, H.T.,Dunlap, R.B.,Lebioda, L.
Catalytic cysteine of thymidylate synthase is activated upon substrate binding.
Biochemistry, 39:6969-6978, 2000

PubMed Abstract: The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.

PubMed: 10841779
DOI: 10.1021/bi000367g

実験手法

X-RAY DIFFRACTION (2.6 Å)