Aldose reductase from human psoas muscle. Affinity labeling of an active site lysine by pyridoxal 5'-phosphate and pyridoxal 5'-diphospho-5'-adenosine.
The reaction of aldose reductase from human psoas muscle with either pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) results in a pseudo first-order 2-fold activation of the enzyme with the stoichiometric incorporation of 1 mol of either reagent per mol of enzyme. However, in addition to an increase in Vmax there was also an increase in Km for both substrate, DL-glyceraldehyde, and coenzyme, NADPH. This resulted in an overall decrease in catalytic efficiency (kcat/Km). Spectral analysis indicated that activation by both PLP and PLP-AMP was accompanied by Schiff's base formation and epsilon-pyridoxyllysine was identified in hydrolysates of the reduced enzyme PLP-complex. Digestion of either PLP-modified or PLP-AMP-modified aldose reductase with endoproteinase Lys-C followed by high performance liquid chromatography purification and amino acid sequencing of the pyridoxyllated peptide revealed that PLP and PLP-AMP had modified the same lysine residue. A 32-residue peptide containing the essential lysine was found to be highly homologous with a segment of the sequence of both human liver aldehyde reductase and rat lens aldose reductase. A tetrapeptide (Ile-Pro-Lys-Ser) containing the essential lysine was identical in all three enzymes. These results highlight the close structural similarity between members of the aldehyde reductase family.
Morjana
N A
NA
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Lyons
C
C
Flynn
T G
TG
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives*; D000345:Affinity Labels / Q000378:metabolism*; D000449:Aldehyde Reductase / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008297:Male; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D013053:Spectrophotometry; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism*
D000345:Affinity Labels; D008024:Ligands; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D013401:Sugar Alcohol Dehydrogenases; D000449:Aldehyde Reductase; C048236:pyridoxal 5'-diphospho-5'-adenosine