fructosyl-glycine and 4-hydroxyphenylglyoxal

Amadoriases I and II are deglycation isoenzymes from Aspergillus sp. of potential relevance for treatment of diabetic complications resulting from excessive protein glycation. Amadoriase II has a preference for anionic substrate with a K(m) of 0.23 and 2.53 mM for fructosylglycine and fructosylpropylamine, respectively. In contrast, the corresponding K(m) values for amadoriase I are 9.75 and 0.023 mM, respectively. Chemical modification of amadoriase II with p-hydroxyphenylglyoxal, a specific arginine-modifying reagent, resulted in an inhibition of enzyme activity toward fructosylglycine, while having less effect on the enzymatic activity toward fructosylpropylamine. Peptide mapping and subsequent mass spectrometry analysis suggest that Arg(112) is one of the sites of p-hydroxyphenylglyoxal modification. Sequence alignment between amadoriase I and amadoriase II revealed that two glutamic acids in amadoriase I align to Arg(112) and Arg(114) in amadoriase II. Site-directed mutation of amadoriase II (R112E, R114E) resulted in reversal of the enzymatic activities toward fructosylglycine and fructosylpropylamine. Our results suggested that Arg(112) and Arg(114) are responsible for the high affinity of amadoriase II toward anionic substrates and determine the substrate selectivity of the enzyme.

Topics: Amino Acid Oxidoreductases; Amino Acid Sequence; Arginine; Aspergillus; Binding Sites; Chromatography, High Pressure Liquid; Fructosamine; Fructose; Glutamic Acid; Glycine; Kinetics; Models, Chemical; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Mapping; Phenylglyoxal; Propylamines; Protein Binding; Recombinant Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Time Factors