Steady-state kinetics of substrate hydrolysis by vacuolar H(+)-pyrophosphatase. A simple three-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 purified enzyme from the same source under a wide range of Mg2+, pyrophosphate (PPi) 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-Mg2+ 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 Mg2+ binding in the absence of PPi is an order of magnitude smaller than that estimated from the steady-state kinetics of PPi hydrolysis. Two distinct Mg(2+)-binding sites are therefore invoked. Since the protective action of Mg2+ is independent of the nature of the monovalent cations and Mg2+ 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 pKa 5.7 and 8.6 in substrate binding and groups of pKa 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.
Baykov
A A
AA
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Bakuleva
N P
NP
Rea
P A
PA
D001665:Binding Sites; D005033:Ethylmaleimide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009994:Osmolar Concentration; D010944:Plants / Q000201:enzymology*; D011188:Potassium / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology*
D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D005033:Ethylmaleimide; D011188:Potassium