Kinetic characterization of the hydrolytic activity of the H+-pyrophosphatase of Rhodospirillum rubrum in membrane-bound and isolated states.
Substrate hydrolysis by the H+-pyrophosphatase (pyrophosphate phosphohydrolase, H+-PPase) of the photosynthetic bacterium Rhodospirillum rubrum follows a two-pathway reaction scheme in which preformed 1:1 and 1:2 . enzyme . Mg2+ complexes (EMg and EMg2) convert dimagnesium pyrophosphate (the substrate). This scheme is applicable to isolated enzyme, uncoupled chromatophores and chromatophores energized by a K+/valinomycin diffusion potential. Tris and other amine buffers exert a specific effect on the bacterial H+-PPase by increasing the Michaelis constant for substrate binding to EMg by a factor of 26-32, while having only small effect on substrate binding to EMg2. Formation of EMg requires a basic group with pKa of 7.2-7.7 and confers resistance against inactivation by mersalyl and N-ethylmaleimide to H+-PPase. The dissociation constants governing EMg and EMg2 formation, as estimated from the mersalyl-protection assays and steady-state kinetics of PPi hydrolysis, respectively, differ by an order of magnitude. Comparison with the data on soluble PPases suggests that, in spite of gross structural differences between H+-PPase and soluble PPases and the added ability of H+-PPase to act as a proton pump, the two classes of enzyme utilize the same reaction mechanism in PPi hydrolysis.
Baykov
A A
AA
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Sergina
N V
NV
Evtushenko
O A
OA
Dubnova
E B
EB
D001426:Bacterial Proteins / Q000187:drug effects / Q000378:metabolism*; D002021:Buffers; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D008566:Membranes / Q000201:enzymology; D008956:Models, Chemical; D011755:Pyrophosphatases / Q000187:drug effects / Q000378:metabolism*; D012247:Rhodospirillum rubrum / Q000201:enzymology*
D001426:Bacterial Proteins; D002021:Buffers; D008565:Membrane Proteins; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D002118:Calcium