Enzymatic activities of rat liver cytosol 10-formyltetrahydrofolate dehydrogenase.
10-Formyltetrahydrofolate dehydrogenase (10-FTH-FDH: EC 1.5.1.6) catalyzes the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) to tetrahydrofolate (H4PteGlu) and CO2 and the NADP(+)-independent hydrolytic cleavage of 10-HCO-H4PteGlu to H4PteGlu and formate. 10-FTHFDH has a 485 amino acid domain at the C-terminus which is 46% identical to aldehyde dehydrogenase (ALDH: EC 1.2.1.3) and contains a conserved active site cysteine (Cys-707). 10-FTHFDH catalyzed NADP(+)-dependent oxidation of propanal and the hydrolysis of p-nitrophenyl acetate (pNPA) in a similar fashion to ALDH. Initial rate studies gave Km values of 46 and 636 microM, respectively, for NADP+ and propanal, while pNPA had a Km of 220 microM. Propanal was able to compete with 10-HCO-H4PteGlu for NADP(+)-dependent oxidation but had no effect on the NADP(+)-independent hydrolase reaction. N-Ethylmaleimide inhibited NADP(+)-dependent 10-HCO-H4PteGlu oxidation but only partially inhibited (65%) hydrolase activity. Disulfiram, a potent inhibitor of cytosolic ALDH, inhibited NADP(+)-dependent propanal oxidation by 10-FTHFDH. We propose that the dehydrogenase reaction of 10-FTHFDH has a mechanism which proceeds through thiohemiacetal and thioester intermediates, similar to that described for aldehyde dehydrogenase. 10-FTHFDH hydrolase activity was dependent on 2-mercaptoethanol and is probably an artifact of the assay system. The N-terminal domain of 10-FTHFDH shows identity to glycinamide ribonucleotide transformylase (EC 2.1.2.2) and contains a putative 10-HCO-H4PteGlu binding site but shows no GAR-TF activity. NADP(+)-dependent oxidation of 10-HCO-H4PteGlu by 10-FTHFDH was inhibited by the folate anti-metabolite, 5,10-dideazatetrahydrofolate, a known GAR-TF inhibitor.
Cook
R J
RJ
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Wagner
C
C
D000447:Aldehydes / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004221:Disulfiram / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D000099295:Formyltetrahydrofolate Dehydrogenase
D000447:Aldehydes; D004221:Disulfiram; D000587:Oxidoreductases Acting on CH-NH Group Donors; D013439:Sulfhydryl Reagents; D000099295:Formyltetrahydrofolate Dehydrogenase; C005556:propionaldehyde