A KINETIC-STUDY OF AN UNSTABLE ENZYME MEASURED THROUGH COUPLING REACTIONS - APPLICATION TO THE SELF-INACTIVATION OF DETERGENT-SOLUBILIZED CA2-ATPASE FROM SARCOPLASMIC-RETICULUM()

A methodology for the kinetic study of the self-inactivation of an uns table enzyme has been developed by using the transient-phase approach when the enzymatic activity is measured through a coupled enzyme syste m. An experimental design has been developed and applied to the inacti vation of the Ca2+-ATPase from sarcoplasmic reticulum solubilized in t he monomeric state. The catalytic activity of the ATP hydrolysis is de termined in the presence of pyruvate kinase and lactate dehydrogenase as auxiliary enzymes, and the oxidation of the last substrate, NADH, i s continuously monitored. The experimental results show that both subs trates, ATP and calcium, protect against enzyme inactivation. This enz yme, the monomeric ATPase, fulfills the catalytic cycle of the native ATPase, and free enzyme and first-calcium bound enzyme are proposed as the intermediates which are being inactivated.