Streptococcus gordonii soluble inorganic pyrophosphatase: an important role for the interdomain region in enzyme activity.
Streptococcus gordonii DL1(Challis) soluble inorganic pyrophosphatase was shown to be a homo dimer with a subunit molecular mass of 33407. In solution, in the presence of Mn(2+), the protein is ellipsoidal with an axial ratio of 3.37 and molecular mass of 67000. In the absence of the divalent cation, the molecular mass is unchanged but the axial ratio increases to 3.94. The enzyme, in the presence of 5 mM Mg(2+), at 25 degrees Celsius and pH 9.0, has K(m) and k(cat) values of 62 microM and 6290 s(-1), respectively. The free N- and C-terminal domains of Streptococcus gordonii PPase did not interact productively when mixed together. Replacing the interdomain region with that from Bacillus subtilis decreased the catalytic efficiency of the enzyme whereas inserting the same region from the Archaeglobus fulgidus thermophilic enzyme yielded an inactive protein. Substitution, deletion and insertion of amino acid residues in the interdomain region were found to affect the monomer dimer equilibrium in the absence of Mn(2+) ions. In the presence of these ions however the variant proteins were dimers. Proteins with altered interdomain regions also displayed a 2- to 625-fold decrease in catalytic efficiency. These data together with that of computer analysis show that the interdomain region has characteristics of a mechanical hinge. Modelling mutant proteins onto the wild type shows that the active site regions are not significantly perturbed. These results show that, although distant from the active site, the interdomain region plays a role in enzyme activity and both its length and composition are important. This supports the hypothesis that catalytic activity requires the N- and C terminal domains of the enzyme to open and close using the interdomain region as a hinge.