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.