Application of the theory of enzyme subunit interactions to ATP-hydrolyzing enzymes. The case of Na,K-ATPase

Abstract

The theory developed by T. L. Hill (1977, Proc. Natl. Acad. Sci. USA, 74:3632-3636) for enzyme interactions is applied to a dimeric enzyme, the subunits of which may each exist in three distinct states (as in a uni-bi kinetic mechanism). It is shown that when simultaneous binding of substrate to both subunits is excluded, the complex kinetic mechanism of the dimer reduces to a simpler scheme with two distinct, but analogous, cycles that are in principle separately observable in kinetic experiments. Because of the intersubunit interactions, which are explicitly taken into account, the two cycles have different Michaelis constants and maximal velocities. The model exhibits negative cooperativity and enhanced reactivity, relative to a monomeric enzyme. The theory is applied to Na,K-ATPase for which a complete, bicyclic, kinetic mechanism and rate constants are available. When taken together with other evidence, structural as well as functional, the striking similarity of the observed kinetics with that developed for a dimeric enzyme strongly suggests that the functional unit of Na,K-ATPase is a dimer. The free energy differences (calculated from the known rate constants) between intermediates are 6-16 kJ/mol, comparable, for example, to the free energy associated with the formation of a base pair in a nucleic acid double helix. The possible relevance of these results for other ATPases is briefly discussed.

2′-O-Dansyl Analogs of ATP Bind with High Affinity to the Low Affinity ATP Site of Na+/K+-ATPase and Reveal the Interaction of Two ATP Sites during Catalysis

“…2, Equation 1). The values of the fitting process agree very well with the published data of other authors (6,8,29). DANS-ATP inhibited competitively the hydrolysis of ATP at both binding sites with positive cooperativity (Figs.…”

“…The bicyclic model of Plesner, on the other hand, gets its power from two ATP binding sites whose partial activities (Na ϩ -ATPase, K ϩ -phosphatase) are lower than the overall reaction (Na ϩ /K ϩ -ATPase). A single subunit does not have to pass all of the intermediates of the Albers-Post circle, but the sum fulfills all steps required for a whole turnover (6). However, there is still a lot of discussion about the intermediates shared by the partial reactions and the overall reaction (7,8).…”

Blocking of Na+/K+ transport by the MgPO4 complex analogue Co(NH3)4PO4 leaves the Na+/Na+‐exchange reaction of the sodium pump unaltered and shifts its high‐affinity ATP‐binding site to a Na+‐like form

“…This is clearly not the case with Co(NH,),PO,, nor with C O ( N H~)~A T P which both bind to the Ez conformation [13,14,161. The simplest explanation of these findings is that Na [32] or variations thereof [33]. A model of this type would not only explain unchanged activities of the El conformation (Figs.…”

Identification, purification and partial characterization of a 70 kDa inhibitor protein of Na+/K+-ATPase from cytosol of pulmonary artery smooth muscle

“…There is a long standing controversy about the minimum catalytic unit of the enzyme Na + /K + -ATPase i.e., whether it exists as αβ protomer or higher oligomers (Repke and Schon 1973;Stein et al 1973;Plesner 1987). Both the αβ protomeric and (αβ) 2 diprotomeric models gained support from various experiments (Ward and Cavieres 1993;Hamer and Schoner 1993).…”