Oxidative modification of aldose reductase induced by copper ion. Definition of the metal-protein interaction mechanism.
Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys(303) appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys(298)-Cys(303). However, a Cys(80)-Cys(303) disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys(80)-Cys(303) disulfide to the more stable product Cys(298)-Cys(303) is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys(80)-Cys(303) disulfide. However, no evidence of conditions favoring the formation of the Cys(298)-Cys(303) disulfide was observed. Our proposal is that the generation of the Cys(298)-Cys(303) disulfide, either directly or by rearrangement of the Cys(80)-Cys(303) disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.
Dipartimento di Fisiologia e Biochimica, Università di Pisa, via S. Maria, 55, Italy.
Ilaria
I
Cecconi
Andrea
A
Scaloni
Giulio
G
Rastelli
Maria
M
Moroni
Pier Giuseppe
PG
Vilardo
Luca
L
Costantino
Mario
M
Cappiello
Donita
D
Garland
Deborah
D
Carper
J Mark
JM
Petrash
Antonella
A
Del Corso
Umberto
U
Mura
D000449:Aldehyde Reductase / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins; D002417:Cattle; D003300:Copper / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D002352:Carrier Proteins; D004220:Disulfides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D003300:Copper; C484670:AKR1B1 protein, human; D000449:Aldehyde Reductase