Open AccessSite specificity of glycation of horse liver alcohol dehydrogenase in vitro
Author(s) -
SHILTON Brian H.,
CAMPBELL Robert L.,
WALTON Donald J.
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb18067.x
Subject(s) - glycation , chemistry , biochemistry , enzyme , nad+ kinase , cofactor , binding site , alcohol dehydrogenase , dehydrogenase , active site , catalysis , aldimine , glucose 6 phosphate dehydrogenase , stereochemistry , receptor
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.