title: Functional role of cysteine-146 in Escherichia coli thymidylate synthase.
abstract: Analysis of mutant Escherichia coli thymidylate synthases (EC 2.1.1.45) with various amino acids substituted for cysteine at position 146 revealed the cysteine to be involved in the binding of 2'-deoxyuridylate as well as initiating the catalytic process. The substitution of a serine or alanine residue at position 146 did not appreciably alter the binding affinity for 2'-deoxyuridylate but the serine mutant enzyme was less active by a factor of 5000, whereas the alanine mutant enzyme was catalytically inactive. In contrast, the substitution of a glycine or threonine at position 146 created inactive enzymes with higher 2'-deoxyuridylate dissociation constants. The dissociation constant values for 2'-deoxyuridylate were used to estimate the overall contribution of the side chain of the amino acid at position 146 to substrate binding. The results suggested that the side chains of cysteine, alanine, and serine make nonspecific but effective van der Waals contacts with 2'-deoxyuridylate, thereby contributing about 0.82 kcal.mol-1 (1 cal = 4.184 J) to the apparent binding energy of the substrate.
authors.affiliation: Department of Microbiology, Wellcome Research Laboratories, Research Triangle Park, NC 27709.
authors.forename: I K
authors.initials: IK
authors.lastname: Dev
authors.forename: B B
authors.initials: BB
authors.lastname: Yates
authors.forename: J
authors.initials: J
authors.lastname: Leong
authors.forename: W S
authors.initials: WS
authors.lastname: Dallas
mesh_terms: D002384:Catalysis; D003545:Cysteine; D004252:DNA Mutational Analysis; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000502:physiology*
chemical_list: D003856:Deoxyuracil Nucleotides; D011624:Pteroylpolyglutamic Acids; D005468:Fluorodeoxyuridylate; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase; D003545:Cysteine; D005998:Glycine