Use of 10-Propargyl-5,8-Dídeazafolate and Directed Mutagenesis to Probe the Catalytic Mechanism of Thymidylate Synthase

Abstract

Thymidylate synthase (TS) catalyzes the conversion of dUMP and CH2H4folate to dTMP and folate by methyl group and hydride transfer. The enzyme has been well characterized from many sources including a 2.1 A resolution X-ray structure of E. coli TS (TS).1 With the exception of TS from protozoans the enzyme consists of two identical subunits with a dimeric molecular weight of approximately 60,000. Although an active site is present on both subunits, there is debate as to whether the active sites function independently, if catalytic activity alternates between subunits, or to what degree of coordination exists between subunits. It is believed that substrate binding and product release are ordered with dUMP binding first, initially to a single subunit, followed by CH2H4folate binding to the same subunit occupied by dUMP.2 Recently, however, it was suggested that the dUMP binding site is accessible in the TS-CH2H4folate-polyglutamate binary complex. 3 These results suggest that TS complexed with the polyglutamated form of CH2H4folate may represent an intermediate along the TS reaction pathway.KeywordsTernary ComplexMutant EnzymeBinary ComplexHydride TransferActive Site CysteineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.