Substrate Channeling and Domain−Domain Interactions in Bifunctional Thymidylate Synthase−Dihydrofolate Reductase

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Abstract
The thymidylate synthase (TS) and dihydrofolate reductase (DHFR) enzymes are found on a single polypeptide chain in several species of protozoa such as the parasitic Leishmania major. Earlier studies with the bifunctional TS−DHFR enzyme from L. major have suggested that this enzyme exhibits a phenomenon known as substrate channeling [Meek, T. D., et al. (1985) Biochemistry24, 678−686]. This is a process by which a metabolite or intermediate is directly transferred from one enzyme active site to the next without being released free into solution. The crystal structure for the bifunctional TS−DHFR enzyme from L. major was recently solved, and it was shown that the TS active site was located 40 Å from the DHFR active site [Knighton, D. R., et al. (1994) Nat.Struct. Biol.1, 186−194]. On the basis of the crystal structure, a novel mechanism has been proposed for the channeling of the intermediate, dihydrofolate, from the TS active site to the DHFR active site [Knighton, D. R., et al. (1994) Nat. Struct. Biol.1, 186−194]. They suggest that the dihydrofolate is transferred via an “electrostatic” channel on the protein surface which connects the two active sites. In this report, we describe the use of a rapid transient kinetic analysis in examining the kinetics of substrate channeling as well as domain−domain interactions in the bifunctional TS−DHFR from L. major.