Navarro-Martínez M D, Cabezas-Herrera J, García-Cánovas F, Rodríguez-López J N
Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Universidad de Murcia, E-301 00 Espinardo, Murcia, Spain.
J Enzyme Inhib Med Chem. 2007 Aug;22(4):377-82. doi: 10.1080/14756360601141653.
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.
尽管诸如甲氧苄啶之类的抗叶酸剂被用于嗜麦芽窄食单胞菌感染的临床治疗，但由于该微生物的二氢叶酸还原酶（DHFR）从未被分离出来，所以对其了解甚少。在此，我们描述了这种酶的纯化过程，并对其受甲氨蝶呤（MTX）抑制的情况进行了动力学表征。经MTX处理后，由于形成了一种寿命长、解离缓慢的酶 - NADPH - 抑制剂复合物，观察到了时间依赖性的慢结合抑制作用。动力学分析揭示了一种一步抑制机制（K（I） = 28.9 +/- 1.9 pM），其缔合速率常数（k（i））为3.8 x 10（7）M（-1）s（-1）。讨论了MTX与嗜麦芽窄食单胞菌DHFR结合的可能机制。
