Specific and reversible inactivation of Phycomyces blakesleeanus isocitrate lyase by ascorbate-iron: role of two redox-active cysteines.
Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe(2+) system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg(2+), isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe(3+) ions produced in a "site-specific" Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.
Rúa
Javier
J
Departamento de Bioquímica y Biología Molecular, Universidad de León, León, 24007, Spain.
Soler
Joaquín
J
Busto
Félix
F
de Arriaga
Dolores
D
D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000378:metabolism* / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004793:Enzyme Reactivators / Q000494:pharmacology; D020868:Gene Silencing / Q000502:physiology*; D007501:Iron / Q000378:metabolism* / Q000494:pharmacology; D007522:Isocitrate Lyase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D010800:Phycomyces / Q000235:genetics / Q000502:physiology*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000494:pharmacology
D000975:Antioxidants; D004791:Enzyme Inhibitors; D004793:Enzyme Reactivators; D013438:Sulfhydryl Compounds; D007501:Iron; D007522:Isocitrate Lyase; D003545:Cysteine; D001205:Ascorbic Acid