Sugars in their open chain conformations are prone to enolization and subsequent autoxidation that yields 1,2 - dicarbonyls capable of covalently modifying proteins and nucleic acids. Superoxide (02 ) both initiates and propagates the oxidation of enediols. Glycation of proteins and subsequent oxidation of the resultant E-Amino fructosyl-lysine residues yields a variety of products, including 1, 2-dicarbonyls. This is believed to be responsible for the deleterious consequences of the hyperglycemia of poorly controlled diabetes mellitus. Our goals are: to further elucidate this deleterious interaction of O2 with sugars using Escherichia coli as our model. We will: isolate, sequence, and clone the defensive glyoxalase III (glo III); create mutants defective in glo III and explore their resistance to short chain sugars and to O2 ; create the glo III mutants in the sod A, sod B background, and in the gsh A background and probe how much the lack of SOD and of GSH exacerbates the glo III deficiency; and to use the superoxide dismutase mimic Mn (III) TM-2-PyP to try to alleviate the consequences of these lacks of glo III. We will explore the substrate specificity of Glo III and compare the kinetic parameters of the different substrates. We will try to find competitive inhibitors of Glo III. We will also explore the reason for the extreme sensitivity of Glo III to inactivation by O2 and by H2O2. What we ,learn with E. coli will certainly illuminate the interactions of O2 and sugars in higher organism.
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