The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His262 has been altered to a tyrosine residue (H262Y-GDH). The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme. Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates. The activation energies for the oxidation of hexoses and pentoses are almost identical. In a previously published model of the enzyme, His262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane. The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected. During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with pK values between pH 7 and pH 8 being involved. Identical results were obtained with H262Y-GDH, showing that His262 it is not directly involved in this process.
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Research Article|
June 08 1999
Characterization of the membrane quinoprotein glucose dehydrogenase from Escherichia coli and characterization of a site-directed mutant in which histidine-262 has been changed to tyrosine
Gyles E. COZIER;
Gyles E. COZIER
1Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Southampton SO16 7PX, U.K.
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Raff A. SALLEH;
Raff A. SALLEH
1Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Southampton SO16 7PX, U.K.
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Christopher ANTHONY
Christopher ANTHONY
1
1To whom correspondence should be addressed (e-mail c.anthony@soton.ac.uk).
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Publisher: Portland Press Ltd
Received:
January 04 1999
Revision Received:
February 18 1999
Accepted:
March 18 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 340 (3): 639–647.
Article history
Received:
January 04 1999
Revision Received:
February 18 1999
Accepted:
March 18 1999
Citation
Gyles E. COZIER, Raff A. SALLEH, Christopher ANTHONY; Characterization of the membrane quinoprotein glucose dehydrogenase from Escherichia coli and characterization of a site-directed mutant in which histidine-262 has been changed to tyrosine. Biochem J 15 June 1999; 340 (3): 639–647. doi: https://doi.org/10.1042/bj3400639
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