Kinetic reaction scheme for the dihydrofolate reductase domain of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major.
In several species of protozoa, the catalytic activities for the enzymes dihydrofolate reductase (DHFR) and thymidylate synthase (TS) reside on a single polypeptide chain constituting a bifunctional thymidylate synthase-dihydrofolate reductase enzyme. In most other species, however, these enzymes occur as monofunctional catalytic activities on separate enzymes. In this study, the kinetic reaction scheme for the dihydrofolate reductase activity from the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) isolated from the parasite Leishmania major is compared to that of the monofunctional DHFR purified from Escherichia coli. Examination using pre-steady-state kinetic methods reveals interesting differences between the bifunctional and monofunctional forms of the dihydrofolate reductase enzymes. The rate-limiting step in the kinetic pathway for the monofunctional E. coli enzyme is the release of product, tetrahydrofolate. In contrast, for the L. major bifunctional enzyme, the kinetic step which limits the steady-state turnover is a conformational change associated with the release of NADP+. A complete kinetic description for the dihydrofolate reductase reaction pathway for the bifunctional enzyme is presented.
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; D005454:Fluorescence Polarization; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D008024:Ligands; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D009097:Multienzyme Complexes; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase