3-phosphohistidine and 6-phospholysine are substrates of a 56-kDa inorganic pyrophosphatase from bovine liver.
A 56-kDa inorganic pyrophosphatase isolated from bovine liver hydrolyzed PPi, imidodiphosphate, 3-phosphohistidine, and 6-phospholysine at rates of 0.11, 0.44, 1.09, and 1.22 mumol/min/mg protein, respectively, in a reaction mixture containing 1 mM MgCl2 at pH 8.2. The hydrolysis of imidodiphosphate was influenced by various treatments in a different manner from that of N-phosphorylated amino acids, indicating that the pyrophosphatase has two different catalytic sites for imidodiphosphate and N-phosphorylated amino acids, respectively. Evidence for separate catalytic sites consists of the following findings: the activity on hydrolysis of imidodiphosphate gave a bell-shaped pH curve with a peak at pH 6.5, while the activity on hydrolysis of N-phosphorylated amino acids maintained a high level in the pH range between 6.0 and 9.5. One hundred micromolar p-chloromercuriphenyl sulfonate inhibited the hydrolysis of imidodiphosphate by 35% and did not inhibit that of N-phosphorylated amino acids. Two millimolar magnesium chloride repressed the hydrolysis of imidodiphosphate and had no inhibitory effect on the hydrolysis of N-phosphorylated amino acids. Moreover, methylenediphosphonic acid, an analog of imidodiphosphate, stimulated the hydrolysis of imidodiphosphate in the presence of MgCl2, while it potentiated the substrate inhibition on hydrolysis of N-phosphorylated amino acids.
Hiraishi
H
H
Department of Biochemistry, Saga Medical School, Japan.
Yokoi
F
F
Kumon
A
A
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D004164:Diphosphonates / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D008970:Molecular Weight; D011755:Pyrophosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D002712:Chlorides; D004164:Diphosphonates; D017895:Manganese Compounds; D015636:Magnesium Chloride; C025308:N-epsilon phospholysine; C016514:imidodiphosphonic acid; D006639:Histidine; D002731:4-Chloromercuribenzenesulfonate; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008239:Lysine; C025340:manganese chloride; C005436:phosphohistidine