A step towards understanding the folding mechanism of horseradish peroxidase

Pappa, Helen S.; Cass, Anthony E. G.

doi:10.1111/j.1432-1033.1993.tb17654.x

Cited by 81 publications

(65 citation statements)

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“…The enzymatic activity of HRP was measured according to the ABTS assay method by following its change in absorbance at 405 nm in 50 mM phosphatekitrate buffer, pH 5.0 (Pappa and Cass, 1993). Absorption measurements were carried out on a…”

Section: Methodsmentioning

confidence: 99%

“…The location of the Trp in the three-dimensional structure has not been idientified due to the absence of an X-ray crystallographic structure, but previous spectroscopic and chemical modification studies indicate that it is not a part of the catalytic site of the enzyme (Brunet et al, 1983;Pappa and Cass, 1993). A very low quantum yield of the tryptophan fluorescence is shwon to be due to intramolecular tryptophan-heme energy transfer in native HRP (Brunet et al, 1983).…”

mentioning

confidence: 99%

“…HRP isoenzyme c by GdmCl have been extensively studied using fluorescence and C D spectroscopies (Pappa and Cass, 1993). However, the principal emphasis of that study lay in following secondary-structural changes of the enzyme and the fate of the heme group at high denaturant concentrations.…”

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confidence: 99%

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Structural Alterations of Horseradish Peroxidase in the Presence of low Concentrations of Guanidinium Chloride

Chakrabarti

Basak

1996

European Journal of Biochemistry

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The presence of very low concentrations of guanidinium chloride (GdmCl) alters the tertiary structure of the monomeric heme-containing enz,yme, horseradish peroxidase (HRP). The change in tertiary structure of the protein was reflected in the mean fluorescence lifetime of its single tryptophan residue, which increased from 2.3 f 0.1 ns in the native enzyme to 2.7 2 0.2 ns in the presence of 100 mM GdmCI. More convincing evidence in support of such alterations came from quenching study of tryptophan fluorescence using the most widely used quencher, acrylamide. It revealed significant differences between the SternVolmer quenching constants observed in the absence and in the presence of GdmCl concentrations below 100 mM. The fluorescence emission maximum of 6-propionyl-2-(dimethylamino)naphthalene (PRO-DAN), used as an extrinsic fluorophore to probe any changes in the tertiary structure of the enzyme, was blue-shifted from 522 nm in aqueous buffer to 509 nm in the presence of 27 pM native HRP. However, this emission maximum appeared at 519 nm when the PRODAN was incorporated in HRP previously incubated with 100 mM GdmC1. The fluorescence lifetime of PRODAN incorporated in HRP was also different from that of PRODAN in buffer, but much more so in absence of GdmCl than in its presence.Taken together, these results indicate partial unfolding of HRP leading to a conformation with native-like secondary structure and unaltered enzymatic activity, in presence of millimolar concentrations of GdmCI.Keywords: horseradish peroxidase; guanidinium chloride; 6-propionyl-2-(dimethylamino)naphthalene; structural alterations ; fluorescence.Guanidinium chloride (GdmC1) has been very frequently used as a strong denaturant of proteins, which have been shown to undergo pronounced structural disintegrations at GdmCl concentrations above 1 M (Tanford, 1968(Tanford, , 1970. However, it is generally accepted that very low concentrations of GdmCl M) which are often present in the buffer during reconstitution of proteins from the completely unfolded state attained by treatment with 4-6 M GdmCI, do not impart any alterations in their tertiary structure. The present experiments are intended to analyze the effect of low concentrations of GdmCl on the tertiary structure of the monomeric enzyme, horseradish peroxidase (HRP) .HRP is a 44-kDa, monomeric heme-protein containing a single tryptophan residue at position 117, making it a good choice for fluorescence spectroscopic studies. The location of the Trp in the three-dimensional structure has not been idientified due to the absence of an X-ray crystallographic structure, but previous spectroscopic and chemical modification studies indicate that it is not a part of the catalytic site of the enzyme (Brunet et al., 1983;Pappa and Cass, 1993). A very low quantum yield of the tryptophan fluorescence is shwon to be due to intramolecular tryptophan-heme energy transfer in native HRP (Brunet et al., 1983). The denaturatiodrenaturation of both holo-HRP and apoCorrespondence to A. Chakrabarti, Biophysics D...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Structural Alterations of Horseradish Peroxidase in the Presence of low Concentrations of Guanidinium Chloride

Chakrabarti

Basak

1996

European Journal of Biochemistry

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“…1. ApoHRPC has been found not to refold properly in the absence of calcium ions (12), and removal of the bound calcium ions from native HRPC results in a considerable decrease in activity and thermal stability (10,11,13,14). These observations demonstrate the essential role played by the structural calcium ions in HRPC in maintaining a heme pocket architecture conducive to high activity.…”

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confidence: 99%

The Critical Role of the Proximal Calcium Ion in the Structural Properties of Horseradish Peroxidase

Howes

Feis

Raimondi³

et al. 2001

Journal of Biological Chemistry

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The extent to which the structural Ca 2؉ ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca 2؉ ion. The Fe(III) heme states are modified upon Ca 2؉ depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca 2؉ -depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca 2؉ depletion are easily reversed. Contrary to the native protein, the Ca 2؉ -depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the (Fe-Im) stretching mode downshifts 27 cm ؊1 upon Ca 2؉ depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca 2؉ -depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca 2؉ depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca 2؉ ion as that which is lost upon Ca 2؉ depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.Peroxidases of the plant peroxidase superfamily are hemecontaining enzymes that oxidize a variety of aromatic molecules in the presence of hydrogen peroxide. They include peroxidases of plant, fungal, and prokaryotic origins that can be divided into three classes based on sequence alignment (1).Additional support for such a classification was gained as the crystal structures of representative enzymes of each class became available. Class I contains bacterial peroxidases and peroxidases from plant mitochondria and chloroplasts, for example most ascorbate peroxidases and cytochrome c peroxidase. Class II contains extracellular fungal peroxidases such as Coprinus cinereus peroxidase (a peroxidase essentially identical to Arthromyces ramosus peroxidase) and lignin-degrading peroxidases. Class III contains secretory plant peroxidases, typified by the classical horseradish peroxidase isoenzyme C (HRPC). The peroxidases of classes II and III share a number of structural elements considered to be of importance for maintaining protein stability and activity. These include calciumbinding sites proximal and distal to the heme and four disulfide bridges (1-5); the latter was in different locations in class II with respect to class III peroxidases. These features are absent in class I peroxidases. Class III peroxidases also have a loop insertion in the sequence, composed of the DЈ, FЈ, and FЉ helices, not found in the other classes. The relationship between calcium binding and enzyme inactivation has been treated primarily by studies on lignin peroxidase (LIP) (6, 7), manganese peroxidase (MNP) (8, 9), and HRPC (10, 11). Thermal (6) and alkaline (7) i...

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“…In certain other cases, although the unfolding curve shows a single step, the transition involves intermediates (2,6,7), which makes the analysis difficult, almost impossible, due to the inability to estimate their fractional contribution to the observed spectroscopic property and thus their concentrations. If, however the number, accumulation, and stability interval of the intermediates are too small, making their presence insignificant, the analysis with the two-state model may provide thermodynamic parameters that can be very close to their true values (6,7).…”

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Thermal Unfolding of Soybean Peroxidase

Kizhakkedathu

Nazeerunnisa

Behere

2002

Journal of Biological Chemistry

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We have earlier reported that both guanidine hydrochloride (GdnHCl)-induced and heat-induced unfolding of seed coat soybean peroxidase (SBP), monitored by far UV CD, show single step transition. However, although GdnHCl-induced unfolding follows a two-state pathway, the heat-induced denaturation proceeds through intermediates as indicated by the very low cooperativity of transition. In the former case, analysis of the data based on the two-state model gives true thermodynamic parameters, whereas underestimated values are obtained in the latter case. Available complex equations also cannot be applied for the analysis of the thermal unfolding of SBP due to the absence of separate transitions for the intermediates. In the present study, we report a method to obtain true thermodynamic parameters from thermal transition curves of SBP using the two-state model. When SBP is subjected to thermal unfolding at high GdnHCl concentrations (5.8 -6.9 M), cooperative behavior is observed, which allowed the analysis by the twostate model to determine their thermodynamic parameters. We then obtained the thermodynamic parameters in the absence of GdnHCl by extrapolating the graph of linear dependence of ⌬H m on T m to the T m corresponding to 0 M GdnHCl. Another key point for checking the validity of our method was the fact that the unfolded state of SBP generated by either heat or GdnHCl is the same by which we could cross-check our results with that obtained from GdnHCl unfolding. Having obtained the true thermodynamic parameters, we report a detailed thermodynamic study of SBP. Further we address the effect of heme in the thermal unfolding mechanism of SBP.Protein folding/unfolding is one of the most intensely studied aspects of modern biophysics and biochemistry. The denaturant-induced unfolding transitions are measured either spectroscopically or calorimetrically. Far UV CD conforms to the secondary structure of a protein and therefore is used as a tool to monitor protein folding/unfolding. The most common denaturing agents used for the unfolding of proteins are heat, chemical denaturants such as urea and guanidine hydrochloride, and extremes of pH. The simplicity of unfolding transition and its reversibility allow a detailed description of its thermodynamics (1). The two-state model (1-3), which assumes that only the native (N) and unfolded (U) states are present at equilibrium (N ª U), seems to be adequate to describe the unfolding curves to determine the thermodynamic parameters accompanying the unfolding of a number of proteins (4, 5). However, in most cases in which the transition is multistep (stable intermediate states), a complete analysis of the system is possible by utilizing more complex equations (2, 3). If the two-state model is still used in such cases, underestimated values for the thermodynamic parameters are obtained (2, 3). In certain other cases, although the unfolding curve shows a single step, the transition involves intermediates (2, 6, 7), which makes the analysis difficult, almost impossible, due to t...

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A step towards understanding the folding mechanism of horseradish peroxidase

Cited by 81 publications

References 35 publications

Structural Alterations of Horseradish Peroxidase in the Presence of low Concentrations of Guanidinium Chloride

Structural Alterations of Horseradish Peroxidase in the Presence of low Concentrations of Guanidinium Chloride

The Critical Role of the Proximal Calcium Ion in the Structural Properties of Horseradish Peroxidase

Thermal Unfolding of Soybean Peroxidase

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