Functional effects of amino acid substitutions at residue 33 of human thymidylate synthase

Archives of Biochemistry and Biophysics
R T ReillyF G Berger

Abstract

Fluorinated pyrimidines, such as 5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd), are cytotoxic to cells as a consequence of generation of 5-fluoro-2'-deoxyuridylate (FdUMP), which is a mechanism-based inhibitor of the enzyme thymidylate synthase (TS). FdUMP inhibits TS via its binding into a stable inhibitory ternary complex (ITC) with the enzyme and the cosubstrate N5, N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4PteGlu). In previous studies, we identified a naturally occurring mutant form of human TS that contains a Tyr-->His substitution at residue 33 and confers relative resistance to FdUrd in both mammalian and bacterial cells. Kinetic studies indicated that the equilibrium dissociation constant (Kd) for binding of FdUMP into the ITC is altered in the mutant enzyme. In the current investigation, we have examined the kinetics of FdUMP binding into covalent binary complexes, i.e., in the absence of CH2H4PteGlu. Our results showed that although the rate constants for binary FdUMP binding (i.e., kon and koff) are altered by the Tyr-->His substitution, there is no measurable effect on the overall Kd. Analysis of a number of other amino acid substitutions at residue 33 indicated that maximal enzyme accumulation and f...Continue Reading

References

Oct 27, 1992·Biochemistry·M PookanjanatavipD V Santi
Dec 1, 1974·Archives of Biochemistry and Biophysics·J L AullR B Dunlap
Nov 1, 1966·Biochemistry·D Roberts
Apr 1, 1980·Archives of Biochemistry and Biophysics·N V BeaudetteR L Kisliuk
Jan 1, 1995·Annual Review of Biochemistry·C W Carreras, D V Santi

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Citations

Aug 28, 2012·Drug Resistance Updates : Reviews and Commentaries in Antimicrobial and Anticancer Chemotherapy·Nitzan Gonen, Yehuda G Assaraf
Dec 23, 2015·Drug Resistance Updates : Reviews and Commentaries in Antimicrobial and Anticancer Chemotherapy·Laura TaddiaMaria Paola Costi
Oct 25, 2003·Oncogene·Rongbao Zhao, I David Goldman
Oct 17, 2007·Journal of Biomolecular Structure & Dynamics·Abul HasnatJulie Andrews
Oct 26, 2021·The FEBS Journal·Sharon SpizzichinoFrancesca Cutruzzolà

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