Substrate channeling and domain-domain interactions in bifunctional thymidylate synthase-dihydrofolate reductase.
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) Biochemistry 24, 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 A 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.
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
P H
PH
Liang
K S
KS
Anderson
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D005454:Fluorescence Polarization; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000737:chemistry / Q000378:metabolism*
D009097:Multienzyme Complexes; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase