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
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;