1evg
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(New page: 200px<br /><applet load="1evg" size="450" color="white" frame="true" align="right" spinBox="true" caption="1evg, resolution 2.00Å" /> '''CRYSTAL STRUCTURE AN...)
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Revision as of 12:13, 20 November 2007
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CRYSTAL STRUCTURE ANALYSIS OF CYS167 MUTANT OF ESCHERICHIA COLI WITH UNMODIFIED CATALYTIC CYSTEINE
Overview
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.
About this Structure
1EVG is a Single protein structure of sequence from Escherichia coli with SO4 as ligand. Active as Thymidylate synthase, with EC number 2.1.1.45 Full crystallographic information is available from OCA.
Reference
Catalytic cysteine of thymidylate synthase is activated upon substrate binding., Phan J, Mahdavian E, Nivens MC, Minor W, Berger S, Spencer HT, Dunlap RB, Lebioda L, Biochemistry. 2000 Jun 13;39(23):6969-78. PMID:10841779
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