title: Identification of the site of non-enzymatic glycation of glutathione peroxidase: rationalization of the glycation-related catalytic alterations on the basis of three-dimensional protein structure.
abstract: Bovine erythrocyte glutathione peroxidase has been glycated in vitro by incubation in 0.05 M glucose at pH 7.4. Upon glycation the estimated KM for t-butylhydroperoxide reduction increased by approx. 3-fold in comparison to non-glycated glutathione peroxidase. The glycated protein fraction was stabilized by NaBH4 reduction and subjected to tryptic cleavage. Affinity chromatography of the tryptic digest on m-aminophenylboronate-Agarose resulted in the isolation of a single glycated peptide. The peptide was identified as T94-K117 by amino-acid composition comparison to the published amino-acid sequence for this enzyme. The glycation site has been identified as the epsilon-NH2 group of K110. Examination of the three-dimensional structure of bovine erythrocyte glutathione peroxidase indicates that K110 lies on the surface of the protein approximately 15 A away from the active site selenocysteine (SEC 45). Modeling studies indicate that K110 can communicate via H-bonded interactions with the alpha-helix containing the active site residues (SEC-45 and R50). The observed elevation of KM upon glycation of bovine glutathione peroxidase is discussed in terms of the disruption of the long range H-bonded interaction.
authors.affiliation: Department of Chemistry and Biochemistry, University of Windsor, Ont., Canada.
authors.forename: J S
authors.initials: JS
authors.lastname: Baldwin
authors.forename: L
authors.initials: L
authors.lastname: Lee
authors.forename: T K
authors.initials: TK
authors.lastname: Leung
authors.forename: A
authors.initials: A
authors.lastname: Muruganandam
authors.forename: B
authors.initials: B
authors.lastname: Mutus
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004912:Erythrocytes / Q000201:enzymology; D005979:Glutathione Peroxidase / Q000737:chemistry*; D006031:Glycosylation; D008958:Models, Molecular; D011487:Protein Conformation; D013379:Substrate Specificity; D014357:Trypsin
chemical_list: D005979:Glutathione Peroxidase; D014357:Trypsin