title: Probing electrostatic channeling in protozoal bifunctional thymidylate synthase-dihydrofolate reductase using site-directed mutagenesis.
abstract: In this study we used site-directed mutagenesis to test the hypothesis that substrate channeling in the bifunctional thymidylate synthase-dihydrofolate reductase enzyme from Leishmania major occurs via electrostatic interactions between the negatively charged dihydrofolate produced at thymidylate synthase and a series of lysine and arginine residues on the surface of the protein. Accordingly, 12 charge reversal or charge neutralization mutants were made, with up to 6 putative channel residues changed at once. The mutants were assessed for impaired channeling using two criteria: a lag in product formation at dihydrofolate reductase and an increase in dihydrofolate accumulation. Surprisingly, none of the mutations produced changes consistent with impaired channeling, so our findings do not support the electrostatic channeling hypothesis. Burst experiments confirmed that the mutants also did not interfere with intermediate formation at thymidylate synthase. One mutant, K282E/R283E, was found to be thymidylate synthase-dead because of an impaired ability to form the covalent enzyme-methylene tetrahydrofolate-deoxyuridate complex prerequisite for chemical catalysis.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Chloé E
authors.initials: CE
authors.lastname: Atreya
authors.forename: Eric F
authors.initials: EF
authors.lastname: Johnson
authors.forename: Jessica
authors.initials: J
authors.lastname: Williamson
authors.forename: Sing-Yang
authors.initials: SY
authors.lastname: Chang
authors.forename: Po-Huang
authors.initials: PH
authors.lastname: Liang
authors.forename: Karen S
authors.initials: KS
authors.lastname: Anderson
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D004795:Enzyme Stability; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D006838:Hydrocarbons; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D008697:Methane / Q000031:analogs & derivatives* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D011799:Quinazolines / Q000378:metabolism; D013053:Spectrophotometry; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D006838:Hydrocarbons; D009097:Multienzyme Complexes; D011799:Quinazolines; D013763:Tetrahydrofolates; C056930:thymidylate synthase-dihydrofolate reductase; D005468:Fluorodeoxyuridylate; C030011:carbene; C010920:dihydrofolate; C030371:5,6,7,8-tetrahydrofolic acid; D009249:NADP; C031662:CB 3717; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D008697:Methane