Specifically targeted modification of human aldose reductase by physiological disulfides.
Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.
Cappiello
M
M
Dipartimento di Fisiologia e Biochimica, Università di Pisa, via S. Maria 55, 56100 Pisa, Italy. cmario@dfb.unipi.it
Voltarelli
M
M
Cecconi
I
I
Vilardo
P G
PG
Dal Monte
M
M
Marini
I
I
Del Corso
A
A
Wilson
D K
DK
Quiocho
F A
FA
Petrash
J M
JM
Mura
U
U
D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D003538:Cystamine / Q000494:pharmacology; D003553:Cystine / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D019803:Glutathione Disulfide; D006801:Humans; D007525:Isoelectric Focusing; D008958:Models, Molecular
D003553:Cystine; D000449:Aldehyde Reductase; D005978:Glutathione; D003538:Cystamine; D019803:Glutathione Disulfide