Mechanistic characterization of Toxoplasma gondii thymidylate synthase (TS-DHFR)-dihydrofolate reductase. Evidence for a TS intermediate and TS half-sites reactivity.
This study describes the use of rapid transient kinetic methods to characterize the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) enzyme from Toxoplasma gondii. In addition to elucidating the detailed kinetic scheme for this enzyme, this work provides the first direct kinetic evidence for the formation of a TS intermediate and for half-sites TS reactivity in human and Escherichia coli monofunctional TS and in T. gondii and Leishmania major bifunctional TS-DHFR. Comparison of the T. gondii TS-DHFR catalytic mechanism to that of the L. major enzyme reveals the mechanistic differences to be predominantly in DHFR activity. Specifically, TS ligand induced domain-domain communication involving DHFR activation is observed only in the L. major enzyme and, whereas both DHFR activities involve a rate-limiting conformational change, the change occurs at different positions along the kinetic pathway.
Department of Pharmacology, School of Medicine, Yale University, New Haven, Connecticut 06520, USA.
Eric F
EF
Johnson
Wolfgang
W
Hinz
Chloe E
CE
Atreya
Frank
F
Maley
Karen S
KS
Anderson
D000818:Animals; D005822:Genetic Vectors; D007700:Kinetics; D008958:Models, Molecular; D008962:Models, Theoretical; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014122:Toxoplasma / Q000201:enzymology*
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase