Characterization of two soluble inorganic pyrophosphatases from Arabidopsis thaliana

Inorganic pyrophosphatases (siPPiase) catalyze the irreversible hydrolysis of pyrophosphate and their function is known to be essential to all living organisms. In this work, the cDNAs coding for two Arabidopsis thaliana siPPiase were obtained from the TAIR initiative and cloned in an expression vector as fusions with gluthathione-S-transferase. The digested pure forms were also similar to each other, both in amino acid sequence, and in kinetic and molecular properties. They showed absolute requirement for Mg2+, and could only be replaced for Mn2+. The only substrate found to be hydrolyzed was pyrophosphate. Their activity was retained during size exclusion chromatography only when eluted with ethylene glycol and Mg2+, and the observed molecular size corresponded to the monomers. The fluorescence of both proteins was consistent with their low tryptophan content, but an unusual fluorescence was found with excitation at 231 nm. The CD spectrum was consistent with a protein rich in antiparallel β sheets and random coils. The pyrophosphate saturation kinetics indicated the ordered binding of free Mg2+ and of the Mg–pyrophosphate complex, which explains an apparent high substrate inhibition found at low levels of Mg2+. Both enzymes showed similar catalytic constants and affinities for the Mg–pyrophosphate complex, while differed in their affinity for free Mg2+. Finally, the available microarray expression data, and the chromatography siPPiase activity profiles are consistent with a housekeeping expression of these enzymes, but the complexity of the profiles suggest a complex role in plant metabolism.