Role of the conserved tryptophan 82 of Lactobacillus casei thymidylate synthase.
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Kealey JT, Eckstein J, Santi DV
Role of the conserved tryptophan 82 of Lactobacillus casei thymidylate synthase.
Chem Biol. 1995 Sep;2(9):609-14.
- PubMed ID
- 9383465 [ View in PubMed]
- 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.