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.

Carnosine-related dipeptides in the mammalian brain

“…This consists of a series of posttranslational modifications in which protein amino groups and side chains react non-enzymatically with monosaccharides (forming the so called 'Amadori products'; Munch et al, 1997). The potential of carnosine to react with sugars is linked to its aminoacid sequence, which is similar to Lys-His, namely the preferred glycation sites in proteins (Shilton and Walton, 1991;Hipkiss et al, 1995). In the process of protein glycosylation, subsequent oxidations and dehydrations lead to the formation of a wide range of products referred to as advanced glycation endproducts (AGEs).…”

Characterization of In Vitro Glycation Sites of Tau

“…However, the glycation potential of a protein depends not only on the lysine content, but also on its structure, which can provide a microenvironment at the sites that can assist glycation (Shapiro et al, 1980;Watkins et al, 1985;Shilton and Walton, 1991;Nacharaju and Acharya, 1992). As tau does not have much of a higher order structure, its primary structure should be the one that determines its glycation potential.…”

“…Glycation of proteins is site-specific, and only a small fraction of lysine residues are modified in a given protein (Shapiro et al, 1980;Watkins et al, 1985;Iberg and Fluckiger, 1986;Shilton and Walton, 1991;Reiger et al, 1992). The influence of glycation of a protein on its structure and function depends upon the sites of that protein glycated.…”

“…c-Amino groups of lysine residues are the potential sites for glycation (Shapiro et al, 1980;Watkins et al, 1985;Iberg and Fluckiger, 1986;Shilton and Walton, 1991;Reiger et al, 1992). In the initial stage of glycation, glucose reacts with amino groups of proteins, forming labile Schiff base adducts.…”

Glycation of antibodies: Modification, methods and potential effects on biological functions

“…[1][2][3][4] It occurs when protein is incubated with reducing sugars, including glucose, galactose 5 and fructose. 6 The reaction between the amino acid and reducing sugar was first described by Maillard in 1912.…”