Substrate Binding to an Active Creatine Kinase with a Thiol-Bound Mercurinitrophenol Chromophoric Probe

Abstract

A mol of creatine kinase (EC 2.7.3.2), an enzyme with two identical subunits can be titrated very rapidly and stoichiometrically with 2 mol of 2-chloromercuri-4-nitrophenol, an environmentally sensitive chromophoric probe specific for thiols. Chemical modification does not inactivate the enzyme nor can the reaction be prevented by a dead-end quaternary complex of enzyme, magnesium, ADP, and creatine with added nitrate. Creatine kinase modified with the chromophoric reagent can be further reacted with 200-fold molar excess of iodoacetamide, resulting in loss of enzymatic activity but without release of bound mercurinitrophenol. The tightly bound chromophore has spectral properties different from free unbound mercurinitrophenol. Only addition of MgADP, or MgATP and creatine, or nitrate, to the modified though active enzyme causes further changes in the visible spectrum of the bound nitrophenol. Addition of nitrate, Mg, ADP, and creatine to the enzyme with bound reporter group, in the order indicated, caused spectral changes; each addition gave a different spectrum.