Inactivation of Phycomyces isocitrate lyase by thiol-reactive reagents. Evidence for an essential thiol group.
Isocitrate lyase from the mycelium of Phycomyces blakesleeanus was inactivated with thiol-reactive reagents, 5,5'-dithiobis-(2-nitrobenzoic)acid, p-hydroxymercuribenzoic acid, N-ethylmaleimide or iodoacetate, at pH 6.8 and 25 degrees C. In all cases the inactivation is characterized by a biphasic kinetic profile. The rapid initial phase of inactivation does not increase linearly with increasing reagent concentration, but exhibits an apparent saturation effect, suggesting the formation of a reversible complex between the enzyme and the reagent prior to the inactivation step. Re-activation of the enzyme was observed under thiol excess treatment. The pH dependence of the initial phase of inactivation suggests that a group on the enzyme with pKa = 6.8 is being modified. The effect of ligands was tested on the inactivation reaction. Mg(2+)-Ds-isocitrate and Ds-isocitrate provided total protection, whereas Mg2+ ions, succinate and oxalate provided only partial protection of the enzyme against inactivation. On the basis of these results, we would suggest that the thiol-reactive reagents modify at least one thiol group crucial for the enzymatic activity and probably located in the interface between succinate and glyoxylate subsite.
Olano
J
J
Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.
de Arriaga
D
D
Rúa
J
J
Busto
F
F
Soler
J
J
D006863:Hydrogen-Ion Concentration; D007522:Isocitrate Lyase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007523:Isocitrates / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010070:Oxalates / Q000378:metabolism; D010800:Phycomyces / Q000201:enzymology*; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D007523:Isocitrates; D010070:Oxalates; D013386:Succinates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C034219:isocitric acid; D019802:Succinic Acid; D007522:Isocitrate Lyase; D008274:Magnesium