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

The l0-formyltetrahydrofolate synthetase (EC 6.3.4.4), 5,10-methenylte trahydrofolate cyclohydrolase (EC 3.5.4.9) and 5,10-methylenetetrahydr ofolate dehydrogenase (EC 1.5.1.5) activities of pea (Pisum sativum L. cv Homesteader) cotyledons were extracted in the presence of phenylme thylsulphonyl 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 purifi ed over 1000-fold by ammonium sulphate precipitation, followed by chro matography on Sephacryl S-300, DEAE-cellulose, Matrex Green A and hydr oxylapatite. Dehydrogenase and cyclohydrolase activities were separate d from synthetase protein at the Matrex Green A step. Synthetase activ ity was associated with a homodimeric protein (subunit M(r) ca 56 000) which strongly cross-reacted with polyclonal antibodies raised agains t homogeneous spinach leaf l0-formyltetrahydrofolate synthetase. This protein lacked NADP- and NAD-dependent 5,10-methylentetrahydrofolate d ehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activity . The affinity of the purified synthetase for (6S)-tetrahydrofolate po lyglutamates increased with the degree of glutamyl conjugation of this substrate (apparent K-m=40 mu M and 3 mu M for the mono- and pentaglu tamate, respectively). The affinities for formate and ATP were also en hanced by (6S)-tetrahydrofolate polyglutamates. The apparent K-m value s 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, respectivel y, when tetrahydrofolate pentaglutamate was provided.