Kinetic characteristics of nucleoside mono-, di- and triphosphatase activities of the periplasmic 5'-nucleotidase of Escherichia coli.
Periplasmic 5'-nucleotidase from Escherichia coli, in addition to the monophosphoesterase activity has a diphosphohydrolase activity, acting on nucleoside di- and triphosphates. We proposed that the monophosphoesterase and diphosphohydrolase activities have their own active site. This proposal is based on the different types of bonds being broken. Chemical modification with selective group reagents did not show differences in the essentiality of some residues, like histidyl, carboxyl and arginyl groups, of these two hydrolytic activities. While kinetic approaches employing the competition plot and unidirectional substrate inhibition point to that diphosphohydrolase activity (ATPase-ADPase) do not share the same active site with monophosphoesterase activity. Western blotting developed with polyclonal anti-placental apyrase antibody revealed a single protein in the periplasmic fraction of 66.5 kDa similar to the Mr of the purified enzyme by isoelectrofocusing.
García
L
L
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile.
Chayet
L
L
Kettlun
A M
AM
Collados
L
L
Chiong
M
M
Traverso-Cori
A
A
Mancilla
M
M
Valenzuela
M A
MA
D015720:5'-Nucleotidase / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001081:Apyrase / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases; D000251:Adenosine Triphosphatases; D001081:Apyrase