Kinetic evidence for cooperative binding of two ortho-phenanthroline molecules to astacus protease during metal removal.
Kinetic evidence is presented that introduces a new possibility for a mechanism of metal removal from a protein by a chelator. Astacus protease is a 22,614 dalton zinc-metalloendopeptidase from the digestive tract of the freshwater crayfish. Recent studies have shown that it contains a single zinc atom and that removal of this metal yields inactive apo-enzyme, which can be reactivated upon readdition of zinc, cobalt, or copper. The enzyme is inactivated by metal chelators in a time and concentration dependent manner. The inactivation of Zn-Astacus protease by 1,10-phenanthroline (OP) can be monitored continuously in the presence of substrate. The concentration of substrate was found to have no effect on the inactivation rate, indicating that the chelator binding during inactivation is of the noncompetitive type. First-order rate constants for the inactivation process are seen to depend on the concentration of chelator in a sigmoidal manner. Based on mathematics analogous to that for cooperativity in enzyme-substrate kinetics, the deduction is made that there are two OP binding sites on the protein and that the rate of inactivation is related to the saturation of both sites with ligand. If one uses this model, the limiting rate constant of inactivation upon saturation of both sites with ligand is 6.76 x 10(-3) sec-1, and the half maximal rate occurs at an OP concentration of 6.52 mM. A mechanism is proposed wherein both protein bound chelators can cooperate during metal removal either by direct chelation of the metal or by allosteric means. The proposed model and the noncompetitive binding of chelator and substrate are discussed in relation to a recently proposed metal binding site.
Wolz
R L
RL
Department of Physiology, University of Heidelberg, West Germany.
Zwilling
R
R
D000818:Animals; D003400:Astacoidea; D007502:Iron Chelating Agents / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008962:Models, Theoretical; D010618:Phenanthrolines / Q000494:pharmacology*; D011485:Protein Binding; D015032:Zinc / Q000378:metabolism
D007502:Iron Chelating Agents; D008024:Ligands; D010618:Phenanthrolines; D008666:Metalloendopeptidases; D015032:Zinc; C025205:1,10-phenanthroline