SITE-SPECIFICITY OF GLYCATION OF HORSE LIVER ALCOHOL-DEHYDROGENASE IN-VITRO

The site specificity of in vitro glycation of horse liver alcohol dehy drogenase (ADH) was examined and the results interpreted in terms Of s tructural features of the enzyme molecule. In a phosphate buffer solut ion, glycation occurred at Lys231 (the main site of glycation in vivo) , at Lys228 (which is not glycated in vivo), and at several unidentifi ed 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, 55 87-5592]. Use of a molecular modelling programme indicated that this c atalyst was likely to be the imidazole group of His348, exerting its e ffect through the hydroxyl of Thr347. Glycation of Lys228 occurred onl y in the presence of phosphate; in this case molecular modelling showe d that the base catalyst could be a phosphate ion, bound to ADH at a p ositive region of the coenzyme binding site. NAD+ inhibited glycation of Lys228 by binding to the enzyme and restricting access to glucose.