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 ba...

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Bibliographic Details
Published in:Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 1997-05, Vol.117 (1), p.135-142
Main Authors: Garcı́a, Lorena, Chayet, Liliana, Kettlun, Ana M., Collados, Lucı́a, Chiong, Mario, Traverso-Cori, Aı́da, Mancilla, Marta, Valenzuela, M.Antonieta
Format: Article
Language:English
Subjects:
5'-Nucleotidase - metabolism
5′-nucleotidase
Adenosine Triphosphatases - metabolism
Apyrase - metabolism
apyrase like-activity
ATP-diphosphohydrolase like-activity
ATPase-ADPase
Binding Sites
Blotting, Western
Cell Membrane - enzymology
Electrophoresis, Polyacrylamide Gel
Enzyme Inhibitors - pharmacology
Escherichia coli
Escherichia coli - enzymology
Hydrolysis
Isoelectric Focusing
Kinetics
Multienzyme Complexes - metabolism
multifunctional enzyme
Nucleoside diphosphohydrolase
periplasmic
Phosphoric Diester Hydrolases - metabolism
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Summary: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.
ISSN:1096-4959
1879-1107
DOI:10.1016/S0305-0491(96)00258-1