An unexpected effect of ATP on the ratio between activity and phosphoenzyme level of Na +/K+-ATPase in steady state

SCHWARZBAUM PJ, KAUFMAN SB, ROSSI RC, GARRAHAN PJ

BIOCHIMICA ET BIOPHYSICA ACTA (ENZIMOLOGY)

Elsevier

Año: 1995 p. 33 - 40

0006-3002

According to the Albers-Post model the hydrolysis of ATP catalyzed by the Na+/K+-ATPase requires the sequential formation of atleast two conformers of a phosphoenzyme (E1P and E2P), followed by the K+-stimulated hydrolysis of E2P. In this paper we show thatthis model is a particular case of a more general class of models in all of which the ratio between ATPase activity (v) and totalphosphoenzyme level (EP) in steady state is determined solely by the rate constants of interconversion between phosphoconformers andof dephosphorylation. Since these are thought to be unaffected by ATP, the substrate curves for ATPase activity and EP should beidentical in shape so that the ratio v/EP ought to be independent of the concentration of ATP. We tested this prediction by parallelmeasurements of v and EP as a function of [ATP] in the absence or presence of non-limiting concentrations of K ÷, Rb ÷ or NH~-. In theabsence of K + or its congeners, both curves followed Michaelis-Menten kinetics, with almost identical K m values (0.16 /zM) so thatv/EP remained independent of [ATP]. In the presence of either K +, Rb + or NH~-, v and EP increased with [ATP] along the sum of twoMichaelis-Menten equations. The biphasic response of v is well known but, to the best of our knowledge, our results are the firstdemonstration that the response of EP to [ATP] is also biphasic. Under these conditions, the ratio v/EP increased with [ATP] from 19.8to 40.1 s -I along a hyperbola that was half-maximal at 9.5 /xM. To preserve the validity of the current model it seems necessary toassume that ATP acts on the EIP ~ E2P transition and/or on the rate of hydrolysis of E2P. The latter possibility was ruled out. We alsofound that to fit the Albers-Post model to our data, the rate constant for K + deocclussion from E 2 has to be about 10-times higher thanthat reported from measurements of partial reactions. The results indicate that the Albers-Post model quantitatively predicts theexperimental behavior of the Na+-ATPase activity but is unable to do this for the Na+/K+-ATPase activity, unless additional and yetunproved hypothesis are included.