Kinetics and mechanism of interaction of 10-propargyl-5,8-dideazafolate with thymidylate synthase.

摘要: The interaction of Lactobacillus casei thymidylate synthase (TS) with 10-propargyl-5,8-dideazafolate (NPQ) in the presence 2'-deoxyuridylate (dUMP) has been investigated. After formation a rapidly reversible dUMP-NPQ-enzyme complex, slow isomerization occurs to provide ternary complex that can be isolated on nitrocellulose membranes or by gel filtration. Unusual features isolable are rate which it is formed (t1/2 = 0.88 h) and at dissociates 26.5 h). complexes contain 2 mol dUMP NPQ bound per dimeric enzyme. Ultraviolet difference spectra dUMP-NPQ-TS shows high wavelength maximum attributed perturbations enzyme and/or ligand chromophores occur upon binding. Data presented suggest involves nucleophilic attack catalytic thiol group 6-position dUMP. Evidence for this as follows: first, there decrease absorbance pyrimidine chromophore 265 nm same complex; second, using [6-3H]dUMP large, inverse alpha-secondary kinetic isotope effect (kappa H/kappa T 0.83) accord sp2 sp3 rehybridization 6-carbon heterocycle. Treatment sodium dodecyl sulfate (NaDodSO4) results dissociation both ligands an unmodified form, consistent proposed structure complex. Isolable also when incubated 5-fluoro-2'-deoxyuridylate (FdUMP) NPQ. Interestingly, FdUMP from these biphasic, one-half nucleotide dissociating exceedingly congruent 100 findings discussed relationship possible use anticancer agent.