ENZYMATIC-ACTIVITIES OF RAT-LIVER CYTOSOL 10-FORMYLTETRAHYDROFOLATE DEHYDROGENASE

10-Formyltetrahydrofolate dehydrogenase (10-FTHFDH: EC 1.5.1.6) catalyzes the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate (10-HCO-H(4)PteGlu) to tetrahydrofolate (H(4)PteGlu) and CO2 and the NADP(+)-independent hydrolytic cleavage of 10-HCO-H(4)PteGlu to H(4)PteGlu 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 K-m values of 46 and 636 mu M, respectively, for NADP(+) and propanal, while pNPA had a K-m of 220 mu M. Propanal was able to compete with 10-HCO-H(4)PteGlu for NADP(+)-dependent oxidation but had no effect on the NADP+-independent hydrolase reaction. N-Ethylmaleimide inhibited NADP(+)-dependent 10-HCO-H(4)PteGlu 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 a-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-H(4)PteGlu binding site but shows no GAR-TF activity, NADP(+)-dependent oxidation of 10-HCO-H(4)PteGlu by 10-FTHFDH was inhibited by the folate anti-metabolite, 5,10-dideazatetrahydrofolate folate, a known GAR-TF inhibitor. (C) 1995 Academic Press, Inc.