Regulation of the cytoplasmic inorganic pyrophosphatase of Rhodospirillum rubrum.

Abstract

This study is concerned with the catalytic and regulatory properties of soluble inorganic pyrophosphatase of the nonsulfur purple photosynthetic bacterium Rhodospirillum rubrum. A procedure is described for reproducible 100‐fold purification of the enzyme, which shows complex kinetic behavior. Free Mg2+ is an activator of the pyrophosphatase, and virtually eliminates the sigmoidicity otherwise observed in the substrate (magnesium pyrophosphate) saturation kinetic curves. In the absence of appreciable free Mg2+, the enzyme is inhibited by 2‐phosphoglyceric acid, which has the effect of increasing the [S]0.5 value (substrate concentration required for half‐maximal velocity) and of decreasing V. The pyrophosphatase, in dilute solution, is slowly inactivated when the pH is less than 8.6, and this is specifically prevented by Zn2+. Inactivation caused by exposure to low pH (7.6–8.2) can be reversed partially by addition of Zn2+ and/or exposure to high pH (9–9.5). Full reactivation can be achieved by proper adjustment of pH and Zn2+ concentration, together with addition of a reductant (dithionite). The pH‐induced inactivation is accelerated by nucleoside triphosphates, reduced pyridine nucleotides, and the allosteric inhibitor, 2‐phosphoglyceric acid. Such inactivations are reversible, and are not accompanied by change in molecular weight of the enzyme (∼100000). It appears that the R. rubrum inorganic pyrophosphatase can exist in several interconvertible states, which are characterized by inherently different specific activities. Transitions between alternative states are influenced by the various chemical modifiers and conditions noted, and the phenomena described provide a basis for further investigation of the mechanisms responsible for regulation of the enzyme's activity.