THE AFFINITY OF PEA COTYLEDON 10-FORMYLTETRAHYDROFOLATE SYNTHETASE FOR POLYGLUTAMATE SUBSTRATES

The l0-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 l0-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 (6S)-tetrahydrofolate polyglutamates increased with the degree of glutamyl conjugation of this substrate (apparent K-m=40 mu M and 3 mu M for the mono- and pentaglutamate, respectively). The affinities for formate and ATP were also enhanced by (6S)-tetrahydrofolate polyglutamates. The apparent K-m values for ATP and formate declined from 94 mu M and 7.6 mM in the presence of tetrahydrofolate monoglutamate to 12 mu M and 35 mu M, respectively, when tetrahydrofolate pentaglutamate was provided.