The multiple activities of polyphosphate kinase of Escherichia coli and their subunit structure determined by radiation target analysis

Abstract

Polyphosphate kinase (PPK), the principal enzyme required for the synthesis of inorganic polyphosphate (polyP) from ATP, also exhibits other enzymatic activities, which differ significantly in their biochemical optima and responses to chemical agents. These several activities include: polyp synthesis (forward reaction), nATP → polyp(n) + nADP (Equation 1); ATP synthesis from polyp (reverse reaction), ADP + polyP(n) → ATP + polyP(n - 1) (Equation 2); general nucleoside-diphosphate kinase, GDP + polyP(n) → GTP + polyP(n - 1) (Equation 3); linear guanosine 5'-tetraphosphate (ppppG) synthesis, GDP + polyP(n) → ppppG + polyP(n - 2) (Equation 4); and autophosphorylation, PPK + ATP → PPK-P + ADP (Equation 5). The Mg2+ optima are 5, 2, 1, and 0.2 nM, respectively, for the activities in Equations 1, 2, 3, and 4. Inorganic pyrophosphate inhibits the activities in Equations 1 and 3 but stimulates that in Equation 4. The kinetics of the activities in Equations 1, 2, and 3 are highly processive, whereas the transfer of a pyrophosphoryl group from polyp to GDP (Equation 4) is distributive and demonstrates a rapid equilibrium, random Bi-Bi catalytic mechanism. Radiation target analysis revealed that the principal functional unit of the homotetrameric PPK is a dimer. Exceptions are a trimer for the synthesis of ppppG (Equation 4) and a tetrameric state for the autophosphorylation of PPK (Equation 5) at low ATP concentrations. Thus, the diverse functions of this enzyme involve different subunit organizations and conformations. The highly conserved homology of PPK among 18 microorganisms was used to determine important residues and conserved regions by alanine substitution, by site- directed mutagenesis, and by deletion mutagenesis. Of 46 single-site mutants, seven exhibit none of the five enzymatic activities; in one mutant, ATP synthesis from polyp is reduced relative to GTP synthesis. Among deletion mutants, some lost all five PPK activities, but others retained partial activity for some reactions but not for others.