Cysteine 707 is involved in the dehydrogenase activity site of rat 10-formyltetrahydrofolate dehydrogenase.
The enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) (EC 1.5.1.6) catalyzes both the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP(+)-independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the 10-FTHFDH (residues 417-902) shows a 46% identity with a series of NAD(+)-dependent aldehyde dehydrogenases (EC 1.2.1.3). All known members of the aldehyde dehydrogenase family and 10-FTHFDH have a strictly conserved cysteine (Cys-707 for 10-FTHFDH), which has been predicted to be at the active site of these enzymes. Rat liver 10-FTHFDH was expressed in a baculovirus system, and site-directed mutagenesis has been used to study the role of cysteine 707 in the activity of 10-FTHFDH. 10-FTHFDH with alanine substituted for cysteine at position 707 had no dehydrogenase activity, while hydrolase activity and binding of NADP+ were unchanged. Light scattering analysis revealed that wild type and mutant 10-FTHFDH exist as tetramers. We conclude that cysteine 707 is directly involved in the active site of 10-FTHFDH responsible for dehydrogenase activity, and there is a separate site for the hydrolase activity.
Krupenko
S A
SA
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee.
Wagner
C
C
Cook
R J
RJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence; D000099295:Formyltetrahydrofolate Dehydrogenase
D003545:Cysteine; D009249:NADP; D000587:Oxidoreductases Acting on CH-NH Group Donors; D000099295:Formyltetrahydrofolate Dehydrogenase