Open AccessKinetic Characterization of the Hydrolytic Activity of the H + ‐Pyrophosphatase of Rhodospirillum rubrum in Membrane‐Bound and Isolated States
Author(s) -
Baykov Alexander A.,
Sergitalja V.,
Evtushenko Olga A.,
Dubnova Elena B.
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00121.x
Subject(s) - rhodospirillum rubrum , mersalyl , inorganic pyrophosphatase , chemistry , pyrophosphatase , pyrophosphate , michaelis–menten kinetics , substrate (aquarium) , stereochemistry , hydrolysis , biochemistry , enzyme , enzyme assay , biology , ecology , mitochondrion
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 · Mg 2+ complexes (EMg and EMg 2 ) 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 EMg 2 . Formation of EMg requires a basic group with p K a of 7.2–7.7 and confers resistance against inactivation by mersalyl and N ‐ethylmaleimide to H + ‐PPase. The dissociation constants governing EMg and EMg 2 formation, as estimated from the mersalyl‐protection assays and steady‐state kinetics of PP i 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 PP i hydrolysis.