Purification and properties of pyrophosphatase of Acinetobacter johnsonii 210A and its involvement in the degradation of polyphosphate.
Inorganic pyrophosphatase (E.C. 3.6.1.1) of Acinetobacter johnsonii 210A was purified 200-fold to apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and triphosphate to orthophosphate. No activity was observed with other polyphosphates and a wide variety of organic phosphate esters. The molecular mass of the enzyme was estimated to be 141 kDa by gelfiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a subunit composition of six identical polypeptides with a molecular mass of 23 kDa. The cation Mg2+ was required for activity, the activity with Mn2+, Co2+ and Zn2+ was 48, 48 and 182% of the activity observed with Mg2+, respectively. The enzyme was heat-stable and inhibited by fluoride and iodoacetamide. The analysis of the kinetic properties of the enzyme revealed an apparent Km for pyrophosphate of 0.26 mM. In A. johnsonii 210A, pyrophosphatase may be involved in the degradation of high-molecular polyphosphates under anaerobic conditions: (i) it catalyses the further hydrolysis of pyrophosphate and triphosphate formed from high-molecular weight polyphosphates by the action of exopolyphosphatase, and (ii) it abolishes the inhibition of polyphosphate: AMP phosphotransferase-mediated degradation by pyrophosphate and triphosphate.
Bonting
C F
CF
Department of Biomolecular Sciences, Agricultural University, Wageningen, The Netherlands.
Gerards
R
R
Zehnder
A J
AJ
Kortstee
G J
GJ
D000150:Acinetobacter / Q000201:enzymology*; D001673:Biodegradation, Environmental; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D043564:Inorganic Pyrophosphatase; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D011122:Polyphosphates / Q000378:metabolism*; D021122:Protein Subunits; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011122:Polyphosphates; D021122:Protein Subunits; D012969:Sodium Fluoride; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D007460:Iodoacetamide