IDENTIFICATION OF THE SITE OF NONENZYMATIC GLYCATION OF GLUTATHIONE-PEROXIDASE - RATIONALIZATION OF THE GLYCATION-RELATED CATALYTIC ALTERATIONS ON THE BASIS OF 3-DIMENSIONAL PROTEIN-STRUCTURE

Bovine erythrocyte glutathione peroxidase has been glycated in vitro b y incubation in 0.05 M glucose at pH 7.4. Upon glycation the estimated K-M for t-butylhydroperoxide reduction increased by approx. 3-fold in comparison to non-glycated glutathione peroxidase. The glycated prote in fraction was stabilized by NaBH4 reduction and subjected to tryptic cleavage. Affinity chromatography of the tryptic digest on m-aminophe nylboronate-Agarose resulted in the isolation of a single glycated pep tide, 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. E xamination of the three-dimensional structure of bovine erythrocyte gl utathione peroxidase indicates that K110 lies on the surface of the pr otein approximate to 15 Angstrom away from the active site selenocyste ine (SEC 45). Modeling studies indicate that K110 can communicate via H-bonded interactions with the alpha-helix containing the active site residues (SEC-35 and R50). The observed elevation of K-M upon glycatio n of bovine glutathione peroxidase is discussed in terms of the disrup tion of the long range H-bonded interaction.