Steady-state kinetic analysis of the Na +/K +-ATPase. The effects of adenosine 5′-[ß, γ-methylene]triphosphate on substrate kinetics

Abstract

We studied the substrate kinetics of the Na+/K+-ATPase in media with adenosine 5'-[beta, gamma-methylene] triphosphate ([beta,gamma-CH2]ATP), an analog of ATP that is resistant to enzymatic hydrolysis. The aim was to analyze from the point of view of steady-state kinetics the mechanism that generates the biphasic response of the Na+/K+-ATPase to ATP. In the absence of K+, the analog acted as a dead-end inhibitor, Kt for this effect was 43.4 muM. In media with K+ and non-saturating concentrations of ATP, [beta, gamma-CH2]ATP stimulated ATPase. With high concentrations of [beta,gamma-CH2]ATP, the response of activity to the concentration of ATP changed from biphasic to hyperbolic. Comparison of these effects with the predictions of reaction mechanisms that display biphasic responses to the substrate showed that: (1) when this response is caused by two independent and non-interacting active sites, an analog of the substrate will not activate but may change the substrate curve from biphasic into hyperbolic, (ii) if there were negative interactions in affinity and positive interactions in reactivity between two active sites, an analog may activate and change the substrate curve from biphasic into hyperbolic, (iii) in models, such as that proposed for the Na+/K+-ATPase by Plesner et al. in 1981 (Blochim. Biophys. Acta 643, 483-494) in which biphasic kinetics is caused by the existence of two reaction cycles for a single active site, the analog will not activate, and (iv) the observed effects of the analog are compatible with models such as that of Albers-Post model and the more recent versions of the Plesner et al. model in which ATP apart from being the substrate is required to accelerate the rate-limiting step of the reaction.