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| | {{STRUCTURE_1evg| PDB=1evg | SCENE= }} | | {{STRUCTURE_1evg| PDB=1evg | SCENE= }} |
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| - | '''CRYSTAL STRUCTURE ANALYSIS OF CYS167 MUTANT OF ESCHERICHIA COLI WITH UNMODIFIED CATALYTIC CYSTEINE'''
| + | ===CRYSTAL STRUCTURE ANALYSIS OF CYS167 MUTANT OF ESCHERICHIA COLI WITH UNMODIFIED CATALYTIC CYSTEINE=== |
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| - | ==Overview==
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| - | 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. | + | The line below this paragraph, {{ABSTRACT_PUBMED_10841779}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 10841779 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_10841779}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Spencer, H T.]] | | [[Category: Spencer, H T.]] |
| | [[Category: Thr167 e. coli thymidylate synthase with unmodified catalytic cysteine]] | | [[Category: Thr167 e. coli thymidylate synthase with unmodified catalytic cysteine]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 15:33:54 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 1 02:04:55 2008'' |
Revision as of 23:04, 30 June 2008
Template:STRUCTURE 1evg
CRYSTAL STRUCTURE ANALYSIS OF CYS167 MUTANT OF ESCHERICHIA COLI WITH UNMODIFIED CATALYTIC CYSTEINE
Template:ABSTRACT PUBMED 10841779
About this Structure
1EVG is a Single protein structure of sequence from Escherichia coli. 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
Page seeded by OCA on Tue Jul 1 02:04:55 2008
