Purification and Properties of Methylene Tetrahydrofolate Reductase from Pig Liver.

Abstract

Methylenetetrahydrofolate reductase has been purified to homogeneity for the first time. Its physical properties (molecular weight of 210,000 (+OR-) 20,000, subunit molecular weight of approximately 75,000, absorbance and fluorescence characteristics of the protein and the FAD prosthetic group, amino acid composition) have been documented. The kinetic parameters of methylenetetrahydrofolate reductase have been examined in the NADPH-menadione oxidoreductase activity, NADPH-methylenetetrahydrofolate oxidoreductase activity, and methyltetrahydrofolate-menadione oxidoreductase activity. The inhibition of the enzyme by S-adenosylmethionine, a negative feedback inhibitor, has been examined, along with the alleviatory effects of S-adenosylhomocysteine on that inhibition. Rapid-reaction reduction with NADPH in the presence and absence of methylenetetrahydrofolate has been documented. Reduction by NADPH has been found to be faster than turnover. Enzyme-monitored turnover experiments yielded a turnover number which agrees with the turnover number obtained by steady-state kinetic analysis. Product inhibition studies were performed on the enzyme, and suggested an ordered sequential kinetic mechanism in which pyridine nucleotide binds first and is released last. A possible mechanism for methylenetetrahydrofolate has been suggested on the basis of these kinetic studies. Inhibition of methylenetetrahydrofolate reductase by quinazolines has been examined. Quinazolines were found to be good inhibitors (K(,i) values in the (mu)molar range). Methylenetetrahydrofolate reductase was found to be situated in the cytosolic compartment of the rat liver cell, along with thymidylate synthase and methionine synthase, suggesting that cellular compartmentalization does not regulate the flow of methylenetetrahydrofolate into the pathways of thymidylate, purine, and methylated metabolite syntheses. The results of the cellular fractionation are compared with the results of other workers.