Mammalian folylpoly-.gamma.-glutamate synthetase. 3. Specificity for folate analogs

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Abstract
A variety of folate analogues were synthesized to explore the specificity of the folate binding site of hog liver folylpolyglutamate synthetase and the requirements for catalysis. Modifications of the internal and terminal glutamate moieties of folate cause large drops in on rates and/or affinity for the protein. The only exceptions are glutamine, homocysteate, and ornithine analogues, indicating a less stringent specificity around the .delta.-carbon of glutamate. It is proposed that initial folate binding to the enzyme involves low-affinity interactions at a pterin and a glutamate site and the first glutamate bound is the internal residue adjacent to the benzoyl group. Processive movement of the polyglutamate chain through the glutamate site and a possible conformational change in the protein when the terminal residue is bound would result in tight binding and would position the .gamma.-carboxyl of the terminal glutamate in the correct position for catalysis. Steric limitations imposed on the internal glutamate residues that loop out and additional steric constraints imposed by binding of different pterin moieties would be expected to effect slight conformational changes in the protein and/or the terminal glutamate and would explain the decrease in on rate and catalytic rate with increased polyglutamate chain length, and the differential effect of one-carbon substitution on the catalytic rate with polyglutamate derivatives. The 4-amino substitution of folate increases the on rate for monoglutamate derivatives but severely impairs catalysis with diglutamate derivatives. Pteroylornithine derivatives are the first potent and specific inhibitors of folylpolyglutamate synthetase to be identified and may act as analogues of reaction intermediates. Other folate derivatives with tetrahedral chemistry replacing the peptide bond, such as pteroyl-.gamma.-glutamyl-[.psi.,CH2-NH]-glutamate, retain affinity for the protein but are considerably less effective inhibitors than the ornithine derivatives.

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