Enhancement of aldose reductase activity by modification of an active site lysine: a possible mechanism for in vivo activation.
Reaction of aldose reductase (ALR2) from pig muscle with pyridoxal 5'-phosphate (pyridoxal-P) and other lysine modifying reagents resulted in activation of the enzyme. The activation by pyridoxal-P showed pH and concentration dependence that was prevented by incubation with NADPH and various cofactor analogues but not by the aldehyde substrate. Spectral analysis of the reaction showed characteristic peaks associated with Schiff's base formation between a lysine amino group and the aldehyde of pyridoxal-P. Subsequent reduction produced spectra characteristic of a phosphopyridoxyllysine bond. Phosphopyridoxyllysine was isolated by amino acid analysis of modified ALR2. Determination of the stoichiometry of bound phosphopyridoxyllysine indicated one mole of pyridoxal-P per mole of enzyme under conditions that produced maximal activation. A single [3H]phosphopyridoxyllysine containing peptide was isolated by high performance liquid chromatography after enzymatic cleavage of the modified enzyme. This 34 residue peptide exhibited considerable sequence homology to the region comprising residues 242 to 275 of human liver ALR1 and a similar region in rat lens ALR2, human muscle ALR2 and human placental ALR2. The activation of ALR2 via formation of a Schiff's base suggests a possible mechanism of activation of the enzyme in vivo by glucose.
Flynn
T G
TG
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Lyons
C
C
Hyndman
D J
DJ
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology*; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism*; D013552:Swine
D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D013401:Sugar Alcohol Dehydrogenases; D000449:Aldehyde Reductase; D008239:Lysine