Nuclear magnetic resonance relaxation studies of the interaction of ligands with the monomer and tetramer forms of formyltetrahydrofolate synthetase.
Previous work using n.m.r. spectroscopy to investigate the binding between formyltetrahydrofolate synthetase and its ligands was done using the catalytically active tetrameric form of the enzyme. By removal of specific monovalent cations the tetramer dissociates to four identical, catalytically inactive monomers, which are capable of binding nucleotides with affinities similar to those obtained with the tetramer. In the studies reported here, we examined the interaction of metal-nucleotide, formate and monovalent cations with the monomer using n.m.r. relaxation measurements. We were able to demonstrate that formate binds to the monomer. The spin-lattice relaxation rate (1/T1) of the formate carbon in the monomer.M2+.ADP.formate complex is enhanced when Mg2+ is replaced by Mn2+. By assuming that the exchange of formate is not rate-limiting and that tau c of the monomer is the same as that of the tetramer, the distance between the Mn2+ and the formate carbon was calculated and found to be similar in the monomer and tetramer complexes. The spin-lattice relaxation rates of [13C]trimethylammonium ion (an inactive monovalent cation), [13C]methylammonium and [15N]ammonium ions (both active monovalent cations), were measured in the presence of tetramer, MnADP and formate. The relaxation rates of methylammonium and ammonium ions were enhanced under these conditions whereas the relaxation rate of trimethylammonium ion was not. The results indicate that the active monovalent cations bind near the MnADP binding site. A distance from the Mn2+ to the ammonium nitrogen of between 0.5 and 0.6 nm was calculated.
Yeh
C H
CH
Department of Biochemistry, University of Kansas, Lawrence 66045.
Hanna
D A
DA
Everett
G W
GW
Himes
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RH
D000641:Ammonia / Q000494:pharmacology; D001665:Binding Sites; D005574:Formate-Tetrahydrofolate Ligase / Q000378:metabolism*; D005561:Formates / Q000378:metabolism; D008024:Ligands; D008025:Ligases / Q000378:metabolism*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008744:Methylamines / Q000378:metabolism
D005561:Formates; D008024:Ligands; D046911:Macromolecular Substances; D008744:Methylamines; C030544:formic acid; D000641:Ammonia; C027451:methylamine; D008025:Ligases; D005574:Formate-Tetrahydrofolate Ligase; C023336:trimethylamine