title: Binding of the 2',5'-ADP subsite stimulates cyclohydrolase activity of human NADP(+)-dependent methylenetetrahydrofolate dehydrogenase/cyclohydrolase.
abstract: The bifunctional dehydrogenase/cyclohydrolase domain of the human trifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase catalyzes two sequential reactions with significant channeling of the intermediate, methenyltetrahydrofolate. Equilibrium dialysis established that a single, high-affinity NADP+ binding site exists per monomer of the dimeric enzyme. Kinetic characterization of NADP+ binding to the dehydrogenase using analogs as inhibitors demonstrated that affinity for this substrate is due almost exclusively to binding at the 2',5'-ADP subsite. The same structural specificities for binding are exhibited by these analogs in their effects on the cyclohydrolase. Both NADP+ and its 3-aminopyridine analog AADP partially inhibit the activity of the cyclohydrolase when assayed with added methenyltetrahydrofolate as substrate. However, under the same conditions, the cyclohydrolase is actually activated by 2',5'-ADP; activation requires the presence of the 5'-phosphate since 2'-AMP binds but does not activate. Nicotinamide ribose monophosphate (NMN) has no detectable effect either alone or in combination with 2',5'-ADP. The results are consistent with the existence of a shared dehydrogenase/cyclohydrolase active site proximal to the 2',5'-ADP subsite. NADP+ reduces the rate of the fully activated cyclohydrolase by 2-fold. Inhibition appears to be due to the loosely bound nicotinamide ring interacting with the common folate subsite, resulting in only partial inhibition by NADP+. The interaction of 2',5'-ADP with the cyclohydrolase suggests a potential role for this portion of the molecule in promoting the efficiency of the channeling of endogenously generated methenyltetrahydrofolate.
authors.affiliation: Department of Biochemistry, McGill University, Montréal, Québec, Canada.
authors.forename: J N
authors.initials: JN
authors.lastname: Pelletier
authors.forename: R E
authors.initials: RE
authors.lastname: MacKenzie
mesh_terms: D000227:Adenine Nucleotides / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000619:Aminohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D045064:Methenyltetrahydrofolate Cyclohydrolase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009249:NADP / Q000378:metabolism / Q000494:pharmacology*; D010658:Phenylglyoxal / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism
chemical_list: D000227:Adenine Nucleotides; D005456:Fluorescent Dyes; D013763:Tetrahydrofolates; D000249:Adenosine Monophosphate; D009249:NADP; D000244:Adenosine Diphosphate; C018092:5,10-methenyltetrahydrofolate; C039995:3-aminopyridine-1,N(6)-ethenoadenine dinucleotide phosphate; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D045064:Methenyltetrahydrofolate Cyclohydrolase; D010658:Phenylglyoxal