84] Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase: A multifunctional protein from porcine liver

Abstract

This chapter discusses the multifunctional protein methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. These enzyme activities have been shown to copurify 100-fold from porcine liver and are now known to comprise the multifunctional proteins in sheep and pig liver, as well as in yeast. The three enzyme assays involve the spectrophotometric measurement of 5,10-methenyltetrahydrofolate produced or hydrolyzed during the incubations. The dehydrogenase activity is determined by acidification of the assay mix after incubation, thereby destroying the nicotinamide adenine dinucleotide phosphate (NADP)H and converting any formyltetrahydrofolate produced by hydrolysis back to the immediate product, methenyltetrahydrofolate. The synthetase is assayed in a similar fashion, where the formyltetrahydrofolate produced, upon incubating enzyme with tetrahydrofolate, adenosine triphosphate (ATP), and formate is converted to 5,10-methenyltetrahydrofolate by acidification. Cyclohydrolase activity is obtained from the rate of hydrolysis of 5,10-methenyltetrahydrofolate monitored as a decrease in absorbance at 355 nm on a recording spectrophotometer. The dehydrogenase and cyclohydrolase activities are found in a 33,000 mol wt portion of the polypeptide, indicating a close physical association. This is further supported by the observation that the product of the dehydrogenase is preferentially channeled through the cyclohydrolase, rather than being released into the medium.