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 Mg(2+) optima are 5, 2, 1, and 0.2 mM, 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.
Highlights
Bonds, is found in all cells in nature [1]
As a NDK, polyP kinase (PPK) catalyzes the transfer of a terminal phosphate residue to ADP or GDP as well as CDP and UDP
The ratio of GTP to ppppG synthesis was changed from 15:1 in the wild type to 25:1 in the mutant. These findings indicate that the catalytic sites of ATP and GTP synthesis are shared
Summary
Biochemical Assays—Purified PPK (100 ng) was added to a reaction buffer (50 mM Hepes (pH 7.5), 50 mM ammonium sulfate, 5 mM MgCl2) at 37 °C, as described previously [2]. To assay for PPK autophosphorylation, 60 –100 ng of an irradiated enzyme was incubated in 50 mM Hepes-KOH (pH 7.2), 40 mM ammonium sulfate, 10 mM MgCl2, and either 5 M or 1 mM [␥-32P]ATP on ice for 5 min; the reaction was terminated by adding 40 mM EDTA. Seven polymerase chain reaction oligonucleotide primers were synthesized: 5Ј-a, CTCGGATCCATGGGTCAGGAAAAG; 5Ј-b, CTCGCATGCATTACGCCGATTTTA; 5Ј-c, CTCGCATGCTTCCGCAATGGTTTT; 3Ј-a, CTCAAGCTTTTATTGAGGTTGTTC; 3Ј-b, GAAGCATGCGGGCAGCCCTTGCTG; 3Ј-c, GAAGCATGCTTTATCAAACCAAAT; and 3Ј-d, GTTGCATGCGTGCTGACGCAGATA with restriction sites added for subcloning into pQE30 These primers were used to generate six deletion mutants (the expected sizes on agarose gels are given in parentheses): PPK⌬135– 687 (0.5 kb), PPK⌬327– 687 (0.98 kb), PPK⌬532– 687 (1.6 kb), PPK⌬1–134 (1.6 kb), PPK⌬135–326 (1.5 kb), and PPK⌬1–326 (1.09 kb), as well as the wild type (2.07 kb). Samples were verified by SDS-PAGE and Western blotting and further characterized by activity assays
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