Tris Is a Competitive Inhibitor of K+ Activation of the Vacuolar H+-Pumping Pyrophosphatase.

The effects of a range of commonly used pH buffers on the hydrolytic activity of the plant vacuolar H+-transporting inorganic pyrophosphatase (V-PPase) from mung bean (Vigna radiata L.) hypocotyls were tested. All of the buffers inhibited K+ stimulation of the V-PPase, and the degree of inhibition was dependent on the concentrations of both the buffer and K+. The effects were dependent on the organic cation used in the buffers, and those tested inhibited in the order: Tris > Bis-Tris-propane > Bicine = Tricine > imidazole. Detailed studies revealed that a model in which Tris affects both the Km and Vmax for K+ stimulation provided an accurate description of the observed kinetics. The ability of different cations to stimulate the V-PPase was measured with a noncompeting buffer (5 mM imidazole-HCl) and the order of effectiveness was K+ = Rb+ > NH4+ >> Cs+ > Na+ > Li+, with the Km for K+ stimulation being about 1 to 2 mM. Published experiments performed in the presence of Tris were re-evaluated and all could be fitted to mixed inhibition kinetics, with kinetic parameters similar to those measured for the mung bean V-PPase. It is concluded that the variations in the published Km for K+ stimulation of the V-PPase are probably due to the effects of pH buffer cations and that the real value for this parameter is in the low millimolar range. The implications of this for regulation of the V-PPase by K+ in vivo and for the role of the enzyme in K+ transport into the vacuole are discussed.