Glycosylation and thermodynamic versus kinetic stability of horseradish peroxidase

Tams, Jeppe Wegener; Welinder, Karen G.

doi:10.1016/s0014-5793(97)01573-1

Cited by 80 publications

(64 citation statements)

References 21 publications

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“…The greater stability of the plant enzyme is probably due to glycosylation [11]. Thermal profiles of His-tagged and non His-tagged wildtype recombinant HRP closely resembled each other (data not shown).…”

Section: Resultsmentioning

confidence: 73%

“…The stability of HRP has a crucial bearing on its applicability and is affected by a number of variables including temperature [8,9,10], calcium content [10], glycosylation [11], pressure [12], irradiation [13] and pH [9,14], along with additional factors such as urea [15,16], guanidinium chloride [15,17] and various solvents including dimethyl sulfoxide [18] and ionic liquids [19]. Directed evolution has been used to alter rHRP thermal stability [20] but we have found no reports of HRP stability manipulation via rational, site-directed mutagenesis.…”

Section: Introductionmentioning

confidence: 99%

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Effects of mutations in the helix G region of horseradish peroxidase

Ryan¹,

Ó’Fágáin²

2008

Biochimie

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Summary.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 V m /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.

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Section: Resultsmentioning

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Effects of mutations in the helix G region of horseradish peroxidase

Ryan¹,

Ó’Fágáin²

2008

Biochimie

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“…Glycosylation appears to have a potential to modulate the physical properties of proteins without compromising enzymatic activity [25,38,[45][46][47][48]. This makes controlled glycosylation an interesting avenue for the engineering and formulation of pharmaceutical peptides and industrial enzymes.…”

Section: Discussionmentioning

confidence: 99%

Glycoprotein-surfactant interactions: A calorimetric and spectroscopic investigation of the phytase-SDS system

Bagger

Hoffmann

Fuglsang

et al. 2007

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 AbstractThe interactions of sodium dodecyl sulfate (SDS) and two glyco-variants of the enzyme phytase from Peniophora lycii were investigated. One variant (Phy) was heavily glycosylated while the other (dgPhy) was enzymatically deglycosylated. Effects at 24°C of titrating SDS to Phy and dgPhy were studied by Isothermal Titration Calorimetry (ITC) and Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. Comparisons of results for the two variants were used to elucidate glycan-surfactant interrelationships.The CD spectra suggested that both the native and the SDS-denatured states of the two variants were mutually similar, and hence that the denaturation process was structurally equivalent for the two glyco-variants.The denatured state was far from fully unfolded and probably retained a substantial content of native-like structure. Furthermore, it was found that the glycans brought about only a small increase in the resistance towards SDS induced denaturation. The SDS concentration required to denature half of the protein molecules differed less than 1 mM for the two variants.The affinity for SDS of both variants was unusually low. The amount of bound SDS (w/w) at different stages of the binding isotherm was 3-10 times lower than that reported for the most previously investigated globular proteins. Analysis of the relative affinity of the glycan and peptide moieties suggested that the carbohydrates bind much less surfactant. At saturation, glycans adsorbed about half as much SDS (in g/g) as the peptide moiety of Phy and about five times less than average proteins.

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“…Wild type recombinant HRP was less stable than the plant enzyme, probably due to glycosylation of the latter [40]. Over a 10-min incubation period, the half-inactivation temperatures for recombinant and plant HRP were 50 o C and 55 o C respectively.…”

Section: Resultsmentioning

confidence: 97%