Mechanism of inhibition of aldose reductase by menadione (vitamin K3).
Incubation of human placental aldose reductase (EC 1.1.1.21) with menadione (0.5-3.0 mM) resulted in time-dependent loss of the catalytic activity of the enzyme. Kinetic analysis of the data suggests that the inactivation process follows a single apparent rate constant that displays hyperbolic dependence on menadione concentration, indicating that menadione forms a kinetically significant, dissociable complex with the enzyme before the formation of an inactive enzyme-menadione complex. The inactivation of the enzyme with menadione was reversed upon dialysis of the inactivated enzyme against buffer containing 10 mM dithiothreitol suggesting that menadione reacts with enzyme sulfhydryl residue(s). Inactivation of the enzyme was significantly prevented by dithiothreitol (5 mM), NADPH (0.1 mM), and DL-glyceraldehyde (10 mM). Correlation of the fractional remaining activity with the extent of modification indicates that loss of catalytic activity corresponds to the modification of a single amino acid residue of the enzyme protein. Recombinant human aldose reductase, obtained by overexpression in Escherichia coli, and aldose reductase in which Cys-80 or Cys-303 was replaced by serine were also inactivated by menadione. However, enzyme in which Cys-298 was replaced by serine was insensitive to menadione. On the basis of these observations, it is suggested that menadione forms a thiodione-like adduct with Cys-298, leading to inactivation of the enzyme.
Bhatnagar
A
A
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Liu
S Q
SQ
Petrash
J M
JM
Srivastava
S K
SK
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D003545:Cysteine / Q000235:genetics; D005260:Female; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D014812:Vitamin K / Q000494:pharmacology*
D011994:Recombinant Proteins; D014812:Vitamin K; D000449:Aldehyde Reductase; D003545:Cysteine