KINETICS OF IRREVERSIBLE INHIBITION OF CREATINE-KINASE DURING MODIFICATION BY O-PHTHALDEHYDE

It has been previously reported that, with a fluorescence probe formed from o-phthaldehyde (OPTA) and the thiol and amino groups at or near the active site of creatine kinase, inactivation and exposure of the p robe take place simultaneously and well before unfolding of the molecu le as a whole. In this study, the inactivation and modification kineti cs of purified rabbit muscle creatine kinase by OPTA have been compare d, the former by following the substrate reaction in the presence of a previously described inactivator. The microscopic rate constants for the reaction of the inactivator with the free enzyme and with the enzy me-substrate complexes were determined. From the results obtained it a ppears that OPTA is noncompetitive with respect to both substrates. Th e inactivation kinetics is monophasic with OPTA, and neither ATP nor c reatine alone affect the rate constant of inactivation of the enzyme, indicating that the irreversible inhibition of creatine kinase by OPTA is of the noncompetitive type.