Inhibition of Stenotrophomonas maltophilia dihydrofolate reductase by methotrexate: a single slow-binding process.
Although antifolates such as trimethoprim are used in the clinical treatment of Stenotrophomonas maltophilia infection, the dihydrofolate reductase (DHFR) of this microorganism is scarcely known because it has never been isolated. Here, we describe the purification of this enzyme and kinetically characterize its inhibition by methotrexate (MTX). Upon MTX treatment, time-dependent, slow-binding inhibition was observed due to the generation of a long-lived, slowly dissociating enzyme-NADPH-inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (K(I) = 28.9 +/- 1.9 pM) with an association rate constant (k(i)) of 3.8 x 10(7) M(-1)s(-1). Possible mechanisms for MTX binding to S. maltophilia DHFR are discussed.
Navarro-Martínez
M D
MD
Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Universidad de Murcia, E-301 00 Espinardo, Murcia, Spain.
Cabezas-Herrera
J
J
García-Cánovas
F
F
Rodríguez-López
J N
JN
D001671:Biochemistry / Q000379:methods; D002626:Chemistry, Pharmaceutical / Q000379:methods; D003460:Crystallization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008727:Methotrexate / Q000737:chemistry / Q000494:pharmacology*; D008956:Models, Chemical; D020615:Stenotrophomonas maltophilia / Q000201:enzymology*; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*
D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate