title: X-ray analysis of structural changes induced by reduced nicotinamide adenine dinucleotide when bound to cysteine-46-carboxymethylated liver alcohol dehydrogenase.
abstract: The structure of the complex between Cys-46-carboxymethylated horse liver alcohol dehydrogenase (CM-LADH) and reduced nicotinamide adenine dinucleotide (NADH) has been determined by X-ray analysis. The complex represents NADH binding to the orthorhombic, "open" conformation of the enzyme. Coenzyme binding here induces a local structural change in the peptide loop 293-297, but there is no domain rotation, as observed for the "closed" conformation of the protein. This local movement of a few residues in the loop is sufficient to trap the nicotinamide ring of NADH within the active-site area close to a productive binding position. The carboxymethyl group on the zinc ligand cysteine-46 is oriented between the pyrophosphate bridge of NADH and the guanidinium group of arginine-369 and can occupy this position because the coenzyme binding cleft remains open and unchanged upon coenzyme binding. The zinc coordination sphere is distorted, and the position of the metal atom is shifted 1 A compared to native unliganded LADH. The distance between the zinc ion and the sulfur of the alkylated cysteine residue is of the order of 3 A. Alkylation experiments were performed at 0.15 and 10 mM iodoacetate, and peptide maps were examined. Gentle treatment with reagent yields an enzyme product which is substituted at only one of the two zinc binding sites per subunit of LADH (Cys-46). This enzyme species maintains its structural integrity; it binds coenzyme which induces conformational changes resolved into two steps. Thus, in addition to the orthorhombic complex, a crystalline NADH complex in the closed conformation of CM-LADH was obtained. These crystals showed enzymic activity, and single crystals were analyzed with microspectrophotometric methods. Formation of the stable crystalline abortive complex between CM-LADH-NAD+ and 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) could be observed upon addition of excess aldehyde to the closed complex of CM-LADH-NADH. The CM-LADH-NAD+-DACA complex is characterized by an intense absorption band with a lambda max at 456 nm which corresponds to a shift in the spectrum of free DACA of approximately 60 nm. At the higher concentration of iodoacetate, three of the cysteine ligands to the second zinc atom (Cys-100, -103, and -111) are alkylated in addition to Cys-46. This enzyme product rapidly denatures and cannot be crystallized under our conditions. This is an experimental indication that the intact noncatalytic zinc binding site contributes to the structural stability of the protein.
authors.forename: E S
authors.initials: ES
authors.lastname: Cedergren-Zeppezauer
authors.forename: I
authors.initials: I
authors.lastname: Andersson
authors.forename: S
authors.initials: S
authors.lastname: Ottonello
authors.forename: E
authors.initials: E
authors.lastname: Bignetti
mesh_terms: D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D003545:Cysteine; D006736:Horses; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D008968:Molecular Conformation; D009243:NAD / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013053:Spectrophotometry; D014961:X-Ray Diffraction
chemical_list: D009243:NAD; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D003545:Cysteine