Steady-state analysis of enzymes with non-Michaelis-Menten kinetics: The transport mechanism of Na + /K + -ATPase.

Procedures to define kinetic mechanisms from catalytic activity measurements that obey the Michaelis-Menten equation are well established. In contrast, analytical tools for enzymes displaying non-Michaelis-Menten kinetics are underdeveloped, and transient-state measurements, when feasible, are therefore preferred in kinetic studies. Of note, transient-state determinations evaluate only partial reactions, and these might not participate in the reaction cycle. Here, we provide a general procedure to characterize kinetic mechanisms from steady-state determinations. We described non-Michaelis-Menten kinetics with equations containing parameters equivalent to k cat and Km and modeled the underlying mechanism by an approach similar to that used under Michaelis-Menten kinetics. The procedure enabled us to evaluate whether Na+ /K+ -ATPase uses the same sites to alternatively transport Na+ and K+ This ping-pong mechanism is supported by transient-state studies but contradicted to date by steady-state analyses claiming that the release of one cationic species as product requires the binding of the other ( ternary-complex mechanism). To derive robust conclusions about the Na+ /K+ -ATPase transport mechanism, we did not rely on ATPase activity measurements alone. During the catalytic cycle, the transported cations become transitorily occluded ( i.e. trapped within the enzyme). We employed radioactive isotopes to quantify occluded cations under steady-state conditions. We replaced K+ with Rb+ because 42 K+ has a short half-life, and previous studies showed that K+ - and Rb+ -occluded reaction intermediates are similar. We derived conclusions regarding the rate of Rb+ deocclusion that were verified by direct measurements. Our results validated the ping-pong mechanism and proved that Rb+ deocclusion is accelerated when Na+ binds to an allosteric, nonspecific site, leading to a 2-fold increase in ATPase activity.