A 5,10-METHYLENETETRAHYDROFOLATE DEHYDROGENASE - 5,10-METHENYLTETRAHYDROFOLATE CYCLOHYDROLASE PROTEIN FROM PISUM-SATIVUM

A cytosolic protein, that exhibits NADP-dependent 5,10-methylenetetrah ydrofolate dehydrogenase (EC 1.5.1.5) and 5,10-methenyltetrahydrofolat e cyclohydrolase (EC 3.5.4.9) activities but lacks 10-formyltetrahydro folate synthetase (EC 6.3.4.3) activity, was isolated from extracts of pea (Pisum sativum L.) cotyledons and leaves. Dehydrogenase: cyclohyd rolase activities co-purified when protein was fractionated by (NH4)(2 )SO4 precipitation, followed by chromatography on Sephacryl S-300, Mat rex Green A and heparin agarose. The resulting protein (M(r) 38 500) w as apparently homogeneous after SDS-polyacrylamide gel electrophoresis and silver staining. Purified enzyme preparations lacked methylenetet rahydrofolate dehydrogenase activity when NAD replaced NADP in the rea ction system. Attempts to sequence this protein suggested that it may be blocked at the N-terminus. Tryptic digestion of the purified protei n resulted in coordinate losses of dehydrogenase and cyclohydrolase ac tivity. The NADP provided significant protection of both enzyme activi ties during short-term treatments with trypsin but additions of 5,10-m ethylenetetrahydrofolate appeared to accentuate the proteolytic loss o f dehydrogenase activity. The apparent Michaelis constants for NADP (1 1 mu M) and methylenetetrahydrofolate (21 mu M) in the dehydrogenase r eaction were not changed significantly when the folylpentaglutamate su bstrate was provided (8 and 25 mu M, respectively). The dehydrogenase and cyclohydrolase activities were competitively inhibited by dihydrof olates. The K-i values of the dehydrogenase reaction indicated that th e pentaglutamate derivative was a more potent inhibitor than dihydrofo late monoglutamate. The ELISA and Western blot analyses, using rabbit polyclonal antibodies raised against the purified enzyme, revealed cro ss-reactivity with proteins of similar molecular size in leaf extracts of wheat, barley, corn, bean and pea. Chromatography of these leaf ex tracts on Matrex Green A, in the presence of protease inhibitors, show ed that 10-formyltetrahydrofolate synthetase was readily separated fro m protein with dehydrogenase and cyclohydrolase activity.