Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide

Ryan, Barry J.; Ó’Fágáin, Ciarán

doi:10.1016/j.biochi.2007.03.013

Cited by 33 publications

(26 citation statements)

References 16 publications

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“…In other instances, Lys and Glu are the objective to be replaced by other less reactive amino acids. For example, horseradish peroxidase (HRP) is a commonly used enzyme in many biotechnological fields, and its low resistance to hydrogen peroxide is a limitation for its implementation in many instances [229]. To reach this goal, 13 single-and three double-mutants of solvent exposed, proximal lysine and glutamic acid residues were analysed.…”

Section: Site Directed Mutagenesismentioning

confidence: 99%

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

Hernández

Berenguer-Murcia²,

Rodrigues³

et al. 2012

COC

140

107

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Hydrogen peroxide is a substrate or side-product in many enzyme-catalyzed reactions. For example, it is a side-product of oxidases, resulting from the re-oxidation of FAD with molecular oxygen, and it is a substrate for peroxidases and other enzymes. However, hydrogen peroxide is able to chemically modify the peptide core of the enzymes it interacts with, and also to produce the oxidation of some cofactors and prostetic groups (e.g., the hemo group). Thus, the development of strategies that may permit to increase the stability of enzymes in the presence of this deleterious reagent is an interesting target. This enhancement in enzyme stability has been attempted following almost all available strategies: site-directed mutagenesis (eliminating the most reactive moieties), medium engineering (using stabilizers), immobilization and chemical modification (trying to generate hydrophobic environments surrounding the enzyme, to confer higher rigidity to the protein or to generate oxidation-resistant groups), or the use of systems capable of decomposing hydrogen peroxide under very mild conditions. If hydrogen peroxide is just a side-product, its immediate removal has been reported to be the best solution. In some cases, when hydrogen peroxide is the substrate and its decomposition is not a sensible solution, researchers coupled one enzyme generating hydrogen peroxide "in situ" to the target enzyme resulting in a continuous supply of this reagent at low concentrations thus preventing enzyme inactivation.This review will focus on the general role of hydrogen peroxide in biocatalysis, the main mechanisms of enzyme inactivation produced by this reactive and the different strategies used to prevent enzyme inactivation caused by this "dangerous liaison". Outlook

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Section: Site Directed Mutagenesismentioning

confidence: 99%

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

Hernández

Berenguer-Murcia²,

Rodrigues³

et al. 2012

COC

140

107

View full text Add to dashboard Cite

show abstract

“…The glycans of HRP appear to belong to the flexible protein surface shielding type [10,11]. The presence of glycans was shown to double the stability of HRP C toward H 2 O 2 [12] and significantly affect its kinetic stability [11]. Lack of post-translational modifications in prokaryotic hosts such as Escherichia coli is the main problem in heterologous expression of such proteins.…”

mentioning

confidence: 98%

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp

Asad

Khajeh

Ghaemi

2010

Appl Biochem Biotechnol

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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 applications. One of the problems with heterologus expression of HRP especially in prokaryotic host is lack of glycosylation that affects it's stability toward H(2)O(2) and thermal inactivation. In this study, two asparagine residues which constitute two of the eight glycosylation sites in native HRP (Asn 13 and 268) with respectively 83% and 65% surface accessibility were substituted with aspartic acid in recombinant HRP. Both mutant proteins expressed in Escherichia coli showed increased stabilities against heat (increase in t (1/2) from 20 min in native rHRP to 32 and 67 min in N13D and N268D) and H(2)O(2) (up to threefold). Unexpectedly, despite the distance of the mutated positions from the active site, notable alterations in steady-state k (cat) and K (m) values occurred with phenol/4-aminoantipyrine as reducing substrate which might be due to conformational changes. No significant alteration in flexibility was detected by acrylamide quenching analyses, but ANS binding experiments purposed lesser binding of ANS to hydrophobic patches in mutated HRPs. Double mutation was non-additive and non-synergistic.

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“…Studies on how the enzymes are inactivated are essential to understanding how to optimize their industrial uses. It is equally important to investigate and describe strategies to minimize inactivation during catalysis [5,26]. The new kinetics parameters defined here would be relevant in the study of mechanism-based inactivation of peroxidases and related systems [27].…”

Section: Inactivation Rate Enhancement Factorsmentioning

confidence: 99%

Mechanism-Based Inhibition of Myeloperoxidase by Hydrogen Peroxide: Enhancement of Inactivation Rate by Organic Donor Substrates

Olorunniji¹,

Iniaghe²,

Adebayo³

et al. 2009

TOEIJ

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Abstract:The effects of two hydrazine derivatives (isoniazid and hydralazine) on the inactivation of myeloperoxidase by H 2 O 2 were investigated. Incubation of 20 nM myeloperoxidase with 0.25 mM H 2 O 2 alone caused a time-dependent irreversible loss of tetramethylbenzidine oxidation activity with a pseudo-first order inactivation rate constant of 0.057 min -1 . The hydrazine derivatives increased the inactivation rate in a concentration-dependent manner. Inactivation of the enzyme by H 2 O 2 with or without the hydrazides showed a saturation kinetics pattern. Steady state kinetics analysis suggests that the hydrazides likely inactivate myeloperoxidase using a similar inactivating species as does H 2 O 2 . A bimolecular rate constant, specific inactivation rate enhancement factor (k* enh ) is proposed as a formal description of the inactivation rate stimulation by the hydrazides. This parameter potentially avoids confounding the finite inactivation due to H 2 O 2 with that caused by the presence of the hydrazides. The relevance of these findings and the constants derived to the analysis of suicide inactivation of peroxidases by reductant substrates are discussed.

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Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide

Cited by 33 publications

References 16 publications

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp

Mechanism-Based Inhibition of Myeloperoxidase by Hydrogen Peroxide: Enhancement of Inactivation Rate by Organic Donor Substrates

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