Studies on the polyglutamate specificity of thymidylate synthase from fetal pig liver

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Abstract
Thymidylate synthase was purified 1700-fold from fetal pig livers by using chromatography on Affigel-Blue, DEAE-52 and hydroxylapatite. Steady-state kinetic measurements indicate that catalysis proceeds via an ordered sequential mechanism. When 5,10-methylenetetrahydropteroylmonoglutamate (CH2-H4PteGlu1) is used as the substrate, dUMP is bound prior to CH2-H4PTeGlu1, and 7,8-dihydropteroylmonoglutamate (H2PteGlu1) is released prior to dTMP. Pteroylpolyglutamates (PteGlun) are inhibitors of thymidylate synthase activity and are competitive with respect to CH2-H4PteGlu1 and uncompetitive with respect to dUMP. Inhibition constants (Ki values), which correspond to dissociation constants for the dissociation of PteGlun from the enzyme-dUMP-PteGlun ternary complex, were determined for PteGlun derivatives with 1-7 glutamyl residues: PteGlu1, 10 .mu.M; PteGlu2, 0.3 .mu.M; PteGlu3, 0.2 .mu.M; PteGlu4, 0.06 .mu.M; PteGlu5, 0.10 .mu.M; PteGlu6, 0.12 .mu.M; PteGlu7, 0.15 .mu.M. Thus, thymidylate synthase preferentially binds pteroylpolyglutamates with 4 glutamyl residues, but derivatives with 2-7 glutamyl residues all bind at least 30-fold more tightly than the monoglutamate. When CH2-H4PteGlu4 is used as the one carbon donor for thymidylate biosynthesis, the order of substrate binding and product release is reversed, with binding of CH2-H4PteGlu4 preceding that of dUMP and release of dTMP preceding release of H2PteGlu4. Vmax and Km values for dUMP and CH2-H4PteGlun show relatively little change as the polyglutamate chain length of the substrate is varied. Comparison of the kinetic data obtained in these studies with earlier studies on methylenetetrahydrofolate reductase from pig liver leads to the prediction that the partitioning of limiting concentrations of CH2-H4PteGlun between the reactions catalyzed by thymidylate synthase and methylenetetrahydrofolate reductase will vary with polyglutamate chain length, with hexaglutamyl substrates preferentially being reduced to methyltetrahydrofolate.

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