A 5,10-methylenetetrahydrofolate dehydrogenase: 5,10-methenyltetrahydrofolate cyclohydrolase protein from Pisum sativum

Abstract

A cytosolic protein, that exhibits NADP-dependent 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) and 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) activities but lacks 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) activity, was isolated from extracts of pea ( Pisum sativum L.) cotyledons and leaves. Dehydrogenase: cyclohydrolase activities co-purified when protein was fractionated by (NH 4) 2SO 4 precipitation, followed by chromatography on Sephacryl S-300, Matrex Green A and heparin agarose. The resulting protein ( M r 38 500) was apparently homogeneous after SDS-polyacrylamide gel electrophoresis and silver staining. Purified enzyme preparations lacked methylenetetrahydrofolate dehydrogenase activity when NAD replaced NADP in the reaction system. Attempts to sequence this protein suggested that it may be blocked at the N-terminus. Tryptic digestion of the purified protein resulted in coordinate losses of dehydrogenase and cyclohydrolase activity. The NADP provided significant protection of both enzyme activities during short-term treatments with trypsin but additions of 5,10-methylenetetrahydrofolate appeared to accentuate the proteolytic loss of dehydrogenase activity. The apparent Michaelis constants for NADP (11 μM) and methylenetetrahydrofolate (21 μM) in the dehydrogenase reaction were not changed significantly when the folylpentaglutamate substrate was provided (8 and 25 μM, respectively). The dehydrogenase and cyclohydrolase activities were competitively inhibited by dihydrofolates. The K i values of the dehydrogenase reaction indicated that the pentaglutamate derivative was a more potent inhibitor than dihydrofolate monoglutamate. The ELISA and Western blot analyses, using rabbit polyclonal antibodies raised against the purified enzyme, revealed cross-reactivity with proteins of similar molecular size in leaf extracts of wheat, barley, corn, bean and pea. Chromatography of these leaf extracts on Matrex Green A, in the presence of protease inhibitors, showed that 10-formyltetrahydrofolate synthetase was readily separated from protein with dehydrogenase and cyclohydrolase activity.