A mechanism for the addition of multiple moles of glutamate by folylpolyglutamate synthetase

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Abstract
The role of the .alpha.-carboxyl group in methotrexate (MeAPA-Glu) and the .gamma.-glutamate derivatives of methotrexate (MeAPA-Glu-Glu) in the reaction catalyzed by folylpolyglutamate synthetase (FPGS) was investigated. MeAPA-Glu and MeAPA-Glu-Glu were accepted as substrates by the same FPGS species contained in an (NH4)2SO4 precipitate of mouse liver protein, as judged by a lack of additivity of product formation at saturating concentrations of both substrates. MeAPA-Gaba, the MeAPA-Glu analog lacking an .alpha.-carboxyl, was inactive as a substrate for this enzyme as was MeAPA-Glu-Gaba, the analog of MeAPA-Glu-Glu that lacked the .alpha.-carboxyl of the terminal glutamic acid. MeAPA-Gaba-Glu, the analog of MeAPA-Glu-Glu without an .alpha.-carboxyl on the first glutamic acid, had activity as a substrate for FPGS that approached that of MeAPA-Glu-Glu. The .alpha.-carboxyl is apparently essential for the binding of folyl monoglutamates to FPGS in the correct orientation to allow catalysis. The binding of the terminal .alpha.-carboxyl of folyl oligoglutamates to the same residue(s) responsible for the binding of the .alpha.-carboxyl of folyl monoglutamates would allow correct positioning of the terminal .gamma.-carboxyl of the chain for reaction. This binding mechanism would be compatible with the utilization of a single enzyme species for the addition of glutamate to the monoglutamate or oligoglutamate forms of folates and folate analogs.

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