The affinity of pea cotyledon 10-formyltetrahydrofolate synthetase for polyglutamate substrates

Abstract The 10-formyltetrahydrofolate synthetase (EC 6.3.4.4), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities of pea ( Pisum sativum L. cv Homesteader) cotyledons were extracted in the presence of phenylmethylsulphonyl fluoride and 25% glycerol to stabilize enzyme activity. These activities were mainly associated with the cytosolic fraction of 1–7 day cotyledons. Synthetase protein of 1 day cotyledons was purified over 1000-fold by ammonium sulphate precipitation, followed by chromatography on Sephacryl S-300, DEAE-cellulose, Matrex Green A and hydroxylapatite. Dehydrogenase and cyclohydrolase activities were separated from synthetase protein at the Matrex Green A step. Synthetase activity was associated with a homodimeric protein (subunit M r ca 56 000) which strongly cross-reacted with polyclonal antibodies raised against homogeneous spinach leaf 10-formyltetrahydrofolate synthetase. This protein lacked NADP- and NAD-dependent 5,10-methylentetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activity. The affinity of the purified synthetase for (6 S )-tetrahydrofolate polyglutamates increased with the degree of glutamyl conjugation of this substrate (apparent K m =40 μM and 3 μM for the mono- and pentaglutamate, respectively). The affinities for formate and ATP were also enhanced by (6 S )-tetrahydrofolate polyglutamates. The apparent K m values for ATP and formate declined from 94 μM and 7.6 mM in the presence of tetrahydrofolate monoglutamate to 12 μM and 35 μM, respectively, when tetrahydrofolate pentaglutamate was provided.