Functional cysteinyl residues in human placental aldose reductase.
Incubation of human placental aldose reductase (EC 1.1.1.21) with the sulfhydryl oxidizing reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) results in a biexponential loss of catalytic activity. Inactivation by DTNB or NEM is prevented by saturating concentrations of NADPH. ATP-ribose offers partial protection against inactivation by DTNB, whereas NADP, nicotinamide mononucleotide (NMN), and the substrates glyceraldehyde and glucose offer little or no protection. The inactivation by DTNB was reversed by dithiothreitol and partially by 2-mercaptoethanol but not by KCN. When the release of 2-nitro-5-mercaptobenzoic acid was measured, 3 mol of sulfhydryl residues was found to be modified per mole of the enzyme by DTNB. Correlation of the fractional activity remaining with the extent of modification by the statistical method of C.-L. Tsou (1962, Sci. Sin. 11, 1535-1558) indicates that of the three reactive residues, one reacts at a faster rate than the other two, and that two residues are essential for the catalytic activity of the enzyme. Labeling of the total sulfhydryl by [14C]NEM and quantification of DTNB-reactive residues in the enzyme denatured by 6 M urea indicates that a total of seven sulfhydryl residues are present in the protein. The modification of the enzyme did not affect Km glyceraldehyde, but the modified enzyme had a lower Km NADPH. Kinetic analysis of the data suggests that a biexponential nature of inactivation could be due to the formation of a dissociable E:DTNB complex and the presence of a partially active enzyme species.
Liu
S Q
SQ
Department of Human Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston 77550.
Bhatnagar
A
A
Das
B
B
Srivastava
S K
SK
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D006801:Humans; D007700:Kinetics; D008962:Models, Theoretical; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011485:Protein Binding; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism*
D004228:Dithionitrobenzoic Acid; D013401:Sugar Alcohol Dehydrogenases; D000449:Aldehyde Reductase; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol