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Effect of polyglutamylation of 5,10-methylenetetrahydrofolate on the binding of 5-fluoro-2'-deoxyuridylate to thymidylate synthase purified from a human colon adenocarcinoma xenografT

Biochemical Pharmacology, ISSN: 0006-2952, Vol: 38, Issue: 2, Page: 335-342
1989
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CH 2 -H 4 PteGlu and H 4 PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of < 3 μM. The interaction of polyglutamates of [6 R ]CH 2,-H 4 PteGlu in the formation and stability of [6- 3 H]FdUMP-thymidylate synthase-CH 2 -H 4 PteGlu n ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6 R ]CH 2 -H 4 PteGlu 3–6 (0.9 to 1.6 μM) were >200-fold and [6 R ]CH 2 -H 4 PteGlu 2 (18.2 μm) was 18-fold more effective than [6 R ]CH 2 -H 4 PteGlu 1 (335 μM) at stabilizing ternary complexes for a T12 for dissociation of 100 min. Polyglutamylation of CH 2 -H 4 PteGlu also increased the affinity of binding of [6- 3 H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 μM, where the K d in the presence of [6 R ]CH 2 -H 4 PteGlu 1 was in the order of 4.0 × 10 −8 M, and for [6 R ]CH 2 -H 4 PteGlu 3–5 was between 3.7 and 5.5 × 10 −9 M. To examine whether this effect was due to differences in the rates at which [6- 3 H]FdUMP was bound ( k on ) or dissociated ( k off ) from the enzyme, the apparent rate of [6- 3 H]FdUMP binding was determined in the presence of [6 R ]CH 2 H 4 PteGlu 1, [6 R ]CH 2 -H 4 PteGlu 3 and [6 R ]CH 2 -H 4 PteGlu 4. The k on values were similar and were in the range of 1.7 to 2.3 × 10 6 M −1 min −1 for 10 or 20 μM folate concentrations. Differences in binding affinity determined for [6 R ]CH 2 -H 4 PteGlu 1 and longer polyglutamate forms of [6 R ]CH 2 -H 4 PteGlu were thus due to differences in k off. The V max for the initial velocity of [6- 3 H]FdUMP binding was achieved at 10 μM folate. Consequently, at concentrations of CH 2 -H 4 PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6 R ]CH 2 -H 4 PteGlu n required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH 2 -H 4 PteGlu n pools to increase the concentrations of the longer polyglutamate species (n ≥ 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H 4 PteGlu.

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