title: Role of the conserved tryptophan 82 of Lactobacillus casei thymidylate synthase.
abstract: BACKGROUND
  Thymidylate synthase (TS; EC 2.1.1.45) catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) by 5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate) to produce 2'-deoxythymidine-5'-monophosphate (dTMP) and 7,8-dihydrofolate (H2folate). Major advances in the understanding of the mechanism of TS have been made by studying site-specific mutants of the enzyme. Trp82 is completely conserved in all of the 20 TS sequences known. It forms part of the CH2H4folate binding pocket, is reported to be a component of a catalytically important H-bond network, and is suspected to be the source of an unusual absorbance change at 330 nm when TS forms a ternary complex with 5-fluoro-dTMP and CH2H4folate. We therefore prepared and characterized a set of 12 mutants at position 82 of Lactobacillus casei TS.
  
  
  RESULTS
  Eight Trp82 mutants were active enough for us to determine their kinetic constants for dTMP production, while four were inactive. The active mutants had higher Km values for dUMP (2- to 10-fold) and CH2H4folate (2- to 27-fold), and lower kcat values (12- to 250-fold) than wild-type TS. The most active mutants were those containing the aromatic side chains Phe and His at position 82. All of the Trp82 mutants catalyzed the debromination of 5-bromo-dUMP with kinetic parameters similar to those of wild-type TS, and all formed ternary complexes with 5-fluoro-dUMP and CH2H4folate. The absence of Trp82 did not prevent the absorbance change at 330 nm on ternary complex formation.
  
  
  CONCLUSIONS
  Trp82, a completely conserved residue that was shown by X-ray crystallography to interact directly with CH2H4folate and indirectly with dUMP, does not appear to be essential for binding or catalysis. We do, however, find a preference for an aromatic side chain at position 82. Trp82 does not contribute to the unique spectral change at 330 nm that accompanies TS ternary complex formation.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California San Francisco 94143-0448, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Kealey
authors.forename: J
authors.initials: J
authors.lastname: Eckstein
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
mesh_terms: D002384:Catalysis; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D005492:Folic Acid / Q000378:metabolism; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D013936:Thymidine / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014364:Tryptophan; D005492:Folic Acid; D013940:Thymidylate Synthase; D013936:Thymidine