Molecular Basis for the Interaction of Polyglutamates of Folic Acid and its Analogs with Dihydrofolate Reductase
Fluorimetric titration has been used to measure the dissociation constants for the complexes of folate, pteroyltriglutamate and pteroylheptaglutamate with dihydrofolate reductase purified from Lactobacillus casei, Streptococcus faecium (isoenzyme 2) and bovine liver. Effects of pH, temperature, salt concentration and second ligands have been examined. The method is shown to be unsuitable for methotrexate complexes.
The polyglutamates do not bind more tightly than folate to the S. faecium reductase under any conditions examined, but bind somewhat more tightly than folate to the L. casei reductase at low pH (<7) and to the bovine liver enzyme at pH 7–9. Increasing concentrations of KCl decrease the binding of all three ligands to the L. casei and bovine liver enzymes. Increasing pH markedly raises the dissociation constants for all complexes of the L. casei reductase, but has only slight effects on the complexes of the S. faecium reductase. Complexes of the bovine enzyme are affected to an intermediate degree by pH, but the folate complex is affected much more than those of the polyglutamates.
Model building studies have been performed with a three-dimensional model of the complex of L. casei reductase with NADPH and methotrexate. Additional glutamyl groups were added in γ-linkage to the glutamate moiety of the complexed methotrexate. A proposed mode of binding of the pteroyl polyglutamates is discussed and sequence comparisons are used to predict residues that might be involved in polyglutamate binding by reductase from other sources.
KeywordsDissociation Constant Dihydrofolate Reductase Bovine Liver Charge Interaction Glutamate Residue
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