STEADY-STATE KINETICS OF SUBSTRATE HYDROLYSIS BY VACUOLAR H-PYROPHOSPHATASE - A SIMPLE 3-STATE MODEL()

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 Vigna radiata (mung bean) and p urified enzyme from the same source under a wide range of Mg2+, pyroph osphate (PP(i)) and K+ concentrations are consistent with a minimal re action scheme in which dimagnesium pyrophosphate is the active substra te species and catalysis is mediated by preformed enzyme-Mg2+ complex. When account is taken of the sensitivity of the V-PPase to ionic stre ngth, additional kinetic interactions are not required to describe the behavior of the enzyme. N-Ethylmaleimide-protection assays show that the dissociation constant for Mg2+ binding in the absence of PP(i) is an order of magnitude smaller than that estimated from the steady-stat e kinetics of PP(i) hydrolysis. Two distinct Mg2+-binding sites are th erefore invoked. Since the protective action of Mg2+ is independent of the nature of the monovalent cation and Mg2+ and K+ do not compete du ring substrate hydrolysis, divalent and monovalent cations are conclud ed to bind at separate sites. The pH dependencies of the kinetic param eters are consistent with the participation of groups of pK(a) 5.7 and 8.6 in substrate binding and groups of pK(a) 6.1 and 9.0 in the 'subs trate-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.