Document Type

Article

Rights

Available under a Creative Commons Attribution Non-Commercial Share Alike 4.0 International Licence

Disciplines

1.6 BIOLOGICAL SCIENCES, Biochemistry and molecular biology, Biochemical research methods

Publication Details

Biochimie, 89, (8), 1029-1032.

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 two-fold) and solvents (up to four-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-sulfonic acid)), despite the distance of the mutated positions from the active site.

DOI

https://doi.org/10.1016/j.biochi.2008.05.008

Funder

Embark Initiative and Dublin City University

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