title: The conformation of NAD+ bound to lactate dehydrogenase determined by nuclear magnetic resonance with suppression of spin diffusion.
abstract: We have reinvestigated the conformation of NAD+ bound to dogfish lactate dehydrogenase (LDH) by using an NMR experiment that allows one to exploit nuclear Overhauser effects to determine internuclear distances between pairs of protons, without perturbation of spin-diffusion effects from other protons belonging either to the cofactor or to the binding pocket of the enzyme. The analysis indicates that the structure of bound NAD+ is in accord with the conformation determined in the solid state by x-ray diffraction for the adenosine moiety, but deviates significantly from that of the nicotinamide. The NMR data indicate conformational averaging about the glycosidic bond of the nicotinamide nucleotide. In view of the strict stereospecificity of catalysis by LDH and the conformational averaging of bound NAD+ that we infer from solution-state NMR, we suggest that LDH binds the cofactor in both syn and anti conformations, but that binding interactions in the syn conformation are not catalytically productive.
authors.affiliation: Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Vincent
authors.forename: C
authors.initials: C
authors.lastname: Zwahlen
authors.forename: C B
authors.initials: CB
authors.lastname: Post
authors.forename: J W
authors.initials: JW
authors.lastname: Burgner
authors.forename: G
authors.initials: G
authors.lastname: Bodenhausen
mesh_terms: D000241:Adenosine / Q000737:chemistry; D000818:Animals; D018360:Crystallography, X-Ray; D004284:Dogfish; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008968:Molecular Conformation; D009243:NAD / Q000737:chemistry*; D009536:Niacinamide / Q000737:chemistry; D012266:Ribose / Q000737:chemistry
chemical_list: D009243:NAD; D009536:Niacinamide; D012266:Ribose; D007770:L-Lactate Dehydrogenase; D000241:Adenosine