Tight-binding complexes of thymidylate synthetase, folate analogs and deoxyribonucleotides.
Tightly-bound ternary complexes are formed when dTMP synthetase is incubated with deoxyribonucleotides such as FdUMP, dUMP, and dTMP, and folate analogs, presumably mediated by conformational changes in the enzyme induced by these ligands. The structure of both the folate analog and the nucleotide determines the tightness of binding in the ternary complex. For example, the first order rate constant for dissociation of FdUMP from the dTMP synthetase-FdUMP PteGlu complex is almost 100-fold larger than from the dTMP synthetase-FdUMP-PteGlu3 complex. The latter had a slower rate of dissociation than did the covalent dTMP synthetase-FdUMP-5,10-CH4H4Pte- Glu complex, which had been shown to have a Kd of 10(-11) M. FdUMP formed tighter complexes than did either dUMP or dTMP when using PteGlu3, PteGlu, or H2PteGlu as the folate components, but with 5,8-deazafolate complexes formed with all three nucleotides appeared to have about the same stability. H2PteGlu also induced the tight binding of dUMP to dTMP synthetase. A Scatchard plot indicated positive cooperativity in the binding of dUMP to both human and bacterial enzyme in the presence of H2PteGlu. These results suggest the possibility that when cells are exposed to MTX the decrease in dTMP synthetase activity may be due largely to formation of inhibitory enzyme-dUMP-H2PteGlun complexes and does not result primarily from depletion of reduced folates because of dihydrofolate reductase inhibition. Because this complex has a slow rate of dissociation, dTMP synthetase activity would still be inhibited for some time after an influx of reduced folates (as in leucovorin rescue) sufficient to support the enzyme reaction under normal conditions.
D000818:Animals; D003854:Deoxyribonucleotides / Q000378:metabolism*; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008780:Methyltransferases / Q000378:metabolism*; D011485:Protein Binding; D013940:Thymidylate Synthase / Q000378:metabolism*
D003854:Deoxyribonucleotides; D005492:Folic Acid; D008780:Methyltransferases; D013940:Thymidylate Synthase