Steady‐state kinetics of substrate hydrolysis by vacuolar H⁺‐pyrophosphatase

Abstract

The results of analyses of the steady-state kinetics of the vacuolar H+-translocating pyrophosphatase (V-PPase) of native tonoplast vesicles isolated from etiolated hypocotyls of fignu rudiutu (mung bean) and purified enzyme from the same source under a wide range of Mg", pyrophosphate (PPJ and K' concentrations are consistent with a minimal reaction scheme in which dimagnesium pyrophosphate is the active substrate species and catalysis is mediated by preformed enzyme-Mg'+ complex. When account is taken of the sensitivity of the V-PPase to ionic strength, additional kinetic interactions are not required to describe the behavior of the enzyme. N-Ethylmaleimide-protection assays show that the dissociation constant for Mg" binding in the absence of PP, is an order of magnitude smaller than that estimated from the steady-state kinetics of PP, hydrolysis. Two distinct Mg"-binding sites are therefore invoked. Since the protective action of Mg2+ is independent of the nature of the monovalent cation and Mg" and K' do not compete during substrate hydrolysis, divalent and monovalent cations are concluded to bind at separate sites. The pH dependencies of the kinetic parameters are consistent with the participation of groups of pK, 5.7 and 8.6 in substrate binding and groups of pK, 6.1 and 9.0 in the substrate-conversion step, indicating that at least four ionizable groups are essential for catalysis. These findings are discussed with respect to the reaction mechanism of the V-PPase and the potential regulatory significance of cytosolic free Mg2+ and K+ in vivo.Plant cells contain alternative metabolic pathways which utilize nucleotides or inorganic pyrophosphate (PP,) as energy sources. While the full significance of this phenomenon remains to be determined, this pattern of alternate proximate energy sources is exemplified by the presence of two parallel H' pumps in the vacuolar membrane (tonoplast). These are the vacuolar H'-ATPase (V-ATPase), an enzyme common to the endomembranes of all characterized eukaryotes (Nelson, 1992;Sze et al., 1992), and a vacuolar H i -translocating inorganic pyrophosphatase (V-PPase), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria (Baltscheffsky, 1978;Rea et al., 1992a;Rea and Poole, 1993). Both enzymes catalyze electrogenic H'-translocation from the cytosol to vacuole lumen to establish an inside-acid pH difference (dpH) and inside-positive electrical potential difference ( d w ) which is employed to energize the H+-coupled, secondary transport of solutes across the vacuolar membrane (Sze, 1985 ;Blumwald, 1987 The potential bioenergetic impact of the V-PPase is indicated by its intrinsic catalytic activity and abundance. The enzyme is capable of generating a steady-state transtonoplast H+-electrochemical potential difference (dPH +) of similar, or greater, magnitude than the V-ATPase on the same membrane (Johannes and Felle, 1989;Maeshima and Yoshida, 1989; Pope and Leigh, 1988;Rea et al., 1992b) and abundance estimates indicate the the V-PPase constitu...

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

“…In this respect, RPPase is similar to the H ϩ -PPase of the plant Vigna radiata (25). The two corresponding pK a values, along with the pHindependent values of the catalytic constant, k h,ind , derived from these dependences with Equation 4 are listed in Table II.…”

“…Calculations and Data Analysis-The amounts of MgCl 2 and PP i required to achieve the desired concentrations of free Mg 2ϩ and the Mg 2 PP i complex in the presence of 50 mM K ϩ were calculated using the apparent dissociation constants for the magnesium and potassium complexes of PP i at pH 7.2 (25) …”

Oxygen exchange reactions catalyzed by vacuolar H+‐translocating pyrophosphatase Evidence for reversible formation of enzyme‐bound pyrophosphate

“…Given the apparent mechanistic conformity of the VPPase and soluble PPases in that in both cases catalysis entails the direct participation of three MgZ+/PPi hydrolyzed [11,27], the finding that the Pc value of the V-PPase for medium PrHOH exchange (0.134). 18) approximates the values obtained for the soluble PPases from Escherichia coli and Saccharomyces cerevisiae [12,26,28,29] indicates that the PP~-P~ interconversion mechanisms of the two classes of enzyme are remarkably alike.…”

“…enzyme-Mg 2÷ complex [11]. Steady-state rate measurements, however, suffer from one major shortcoming: they do not enable resolution of the catalytic steps following substrate binding.…”

“…Second, the pronounced Mg 2+ concentration dependence is in agreement with what is known of the substrate hydrolytic activity of the V-PPase. Since the hydrolysis of PPi requires the formation of an MgzPPi complex [11], it is reasonable that binding of two MgPi molecules will be required for the reaction resulting in medium P~-HOH oxygen exchange. Third, unlike soluble PPases, whose activity is not specifically modulated by monovalent cations, the V-PPase is strongly stimulated by these ions in the sequence K ÷ > Na ÷ > none.…”