Kinetics and mechanism of interaction of 10-propargyl-5,8-dideazafolate with thymidylate synthase
Abstract
The interaction of Lactobacillus casei thymidylate synthase (TS) with 10-propargyl-5,8-dideazafolate (NPQ) in the presence of 2'-deoxyuridylate (dUMP) has been investigated. After formation of a rapidly reversible dUMP-NPQ-enzyme complex, a slow isomerization occurs to provide a ternary complex that can be isolated on nitrocellulose membranes or by gel filtration. Unusual features of the isolable complex are the slow rate by which it is formed (t1/2 = 0.88 h) and the slow rate at which it dissociates (t1/2 = 26.5 h). The ternary complexes contain 2 mol of dUMP and 2 mol of NPQ bound per mol of dimeric enzyme. Ultraviolet difference spectra of the dUMP-NPQ-TS complex shows a high wavelength maximum that has been attributed to perturbations of the enzyme and/or ligand chromophores that occur upon binding. Data are presented that suggest that the formation of the isolable ternary complex involves nucleophilic attack by a catalytic thiol group of the enzyme to the 6-position of dUMP. Evidence for this is as follows: first, there is a decrease in the absorbance of the pyrimidine chromophore at 265 nm that occurs at the same rate as the formation of the isolable complex; second, using [6-3H]dUMP there is a large, inverse alpha-second...Continue Reading
Citations
Distribution of mutations in human thymidylate synthase yielding resistance to 5-fluorodeoxyuridine.
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