title: Site specificity of glycation of horse liver alcohol dehydrogenase in vitro.
abstract: The site specificity of in vitro glycation of horse liver alcohol dehydrogenase (ADH) was examined and the results interpreted in terms of structural features of the enzyme molecule. In a phosphate buffer solution, glycation occurred at Lys231 (the main site of glycation in vivo), at Lys228 (which is not glycated in vivo), and at several unidentified positions. Buffer anions or NAD+ did not affect glycation of Lys231; this supported our hypothesis that the base catalyst which removes a proton from carbon 2 of a Lys231-attached aldimine is part of the ADH molecule [Shilton, B.H. & Walton, D.J. (1991) J. Biol. Chem. 266, 5587-5592]. Use of a molecular modelling programme indicated that this catalyst was likely to be the imidazole group of His348, exerting its effect through the hydroxyl of Thr347. Glycation of Lys228 occurred only in the presence of phosphate; in this case molecular modelling showed that the base catalyst could be a phosphate ion, bound to ADH at a positive region of the coenzyme binding site. NAD+ inhibited glycation of Lys228 by binding to the enzyme and restricting access to glucose.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Canada.
authors.forename: B H
authors.initials: BH
authors.lastname: Shilton
authors.forename: R L
authors.initials: RL
authors.lastname: Campbell
authors.forename: D J
authors.initials: DJ
authors.lastname: Walton
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D005947:Glucose / Q000378:metabolism*; D006736:Horses; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity; D013431:Sulfates / Q000494:pharmacology
chemical_list: D010710:Phosphates; D012492:Salts; D013431:Sulfates; D009243:NAD; C012036:sodium sulfate; D000426:Alcohol Dehydrogenase; D005947:Glucose; C018279:sodium phosphate