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Effects of mutations in the helix G region of horseradish peroxidase

Ryan, Barry J. and Ó'Fágáin, Ciarán (2008) Effects of mutations in the helix G region of horseradish peroxidase. Biochimie, 90 (9). pp. 1414-1421. ISSN 0300-9084

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Abstract

Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its usefulness. Based on prior art, we substituted solvent-exposed lysine and glutamic acid residues near the proximal helix G (Lys 232, 241; Glu 238, 239) and between helices F and F′ (Lys 174). Three single mutants (K232N, K232F, K241N) demonstrated increased stabilities against heat (up to 2-fold) and solvents (up to 4-fold). Stability gains are likely due to improved hydrogen bonding and space-fill characteristics introduced by the relevant substitution. Two double mutants showed stability gains but most double mutations were non-additive and non-synergistic. Substitutions of Lys 174 or Glu 238 were destabilising. Unexpectedly, notable alterations in steady-state Vm/E values occurred with reducing substrate ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), despite the distance of the mutated positions from the active site.

Item Type:Article (Published)
Refereed:Yes
Uncontrolled Keywords:Recombinant; Horseradish peroxidase; Site-specific mutagenesis; Protein stabilisation;
Subjects:Biological Sciences > Biotechnology
Biological Sciences > Enzymology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > National Centre for Sensor Research (NCSR)
Publisher:Elsevier
Official URL:http://dx.doi.org/10.1016/j.biochi.2008.05.008
Copyright Information:Copyright © 2008 Elsevier Masson SAS
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 License. View License
ID Code:541
Deposited On:02 Sep 2008 17:24 by DORAS Administrator. Last Modified 16 Feb 2009 16:00

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