Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 33539-33544 |
| Number of pages | 6 |
| Journal | Journal of Biological Chemistry |
| Volume | 271 |
| Issue number | 52 |
| DOIs | |
| State | Published - 1996 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Specifically targeted modification of human aldose reductase by physiological disulfides. / Cappiello, Mario; Voltarelli, Margaret; Cecconi, Ilaria; Vilardo, Pier Giuseppe; Dal Monte, Massimo; Marini, Isabella; Del Corso, Antonella; Wilson, David K.; Quiocho, Florante A.; Mark Petrash, J.; Mura, Umberto.
In: Journal of Biological Chemistry, Vol. 271, No. 52, 1996, p. 33539-33544.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Specifically targeted modification of human aldose reductase by physiological disulfides
AU - Cappiello, Mario
AU - Voltarelli, Margaret
AU - Cecconi, Ilaria
AU - Vilardo, Pier Giuseppe
AU - Dal Monte, Massimo
AU - Marini, Isabella
AU - Del Corso, Antonella
AU - Wilson, David K.
AU - Quiocho, Florante A.
AU - Mark Petrash, J.
AU - Mura, Umberto
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=12644291191&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=12644291191&partnerID=8YFLogxK
U2 - 10.1074/jbc.271.52.33539
DO - 10.1074/jbc.271.52.33539
M3 - Article
C2 - 8969219
AN - SCOPUS:12644291191
VL - 271
SP - 33539
EP - 33544
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 52
ER -