Calcium-related properties of horseradish peroxidase

Haschke, Richard H.; Friedhoff, Jacqueline M.

doi:10.1016/0006-291x(78)91350-5

Cited by 136 publications

(71 citation statements)

References 3 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…HRP has been reported to contain two bound Ca", which are important for both the activity and thermostability of the protein (Haaschke and Friedhoff, 1978). NMR experiments Each sample was excited at 398 nm and the fluorescence intensity at 620 nm was measured.…”

Section: Stabilising Effect Of Ca2+ On the Hrpmentioning

confidence: 99%

A step towards understanding the folding mechanism of horseradish peroxidase

Pappa

Cass

1993

European Journal of Biochemistry

View full text Add to dashboard Cite

The guanidinium chloride denaturatiodrenaturation of the holo-and apo-horseradish peroxidase isoenzyme c (HRP) has been studied by fluorescence and circular dichroism spectroscopies.A distinct equilibrium intermediate of the apoprotein could be detected at low concentrations of guanidinium chloride (0.5 M). This intermediate has a secondary structure content like that of the native protein but a poorly defined tertiary structure. Renaturation of the apo-HRP is reversible and 100% activity could be obtained after addition of a twofold excess of free haem. The denaturation of the holo-HRP is more complex and occurs in two distinct steps; unfolding of the protein backbone and loss of the haem. The denatured protein folds back to its native conformation but the incorporation of the haem occurs only after the secondary structure is formed.Ca2+ appears to be important for the stability of the protein as the apo-HRP is more resistant to denaturation in the presence of Ca". The free-energy change during unfolding of the apo-HRP was determined in the absence and presence of Ca" and found to be 9.2 kJ/mol and 16.7 kJ/mol, respectively. The importance of Caz+ to the protein stability was also supported by studies on the loss of the haem from the protoporphyrin-IX-apo-HRP complex.Horseradish peroxidase (HRP) has been extensively investigated over many years and has found widespread application in colorimetric test strips, as a label in both antibody and DNA probe methods and as a component of enzyme electrodes. In the horseradish root, HRP is found as a family of isoenzymes which appear to be the result of both multiple genes (Fujiyama et al., 1988) and different degrees of posttranslational modification with carbohydrate (Aibara et al., 1981). The most abundant isoenzyme is HRP C, which has a molecular mass of 44 kDa, a known amino acid sequence of 308 residues (Welinder, 1979) and, in addition to the iron (111) protoporphyrin IX active centre, has eight N-linked carbohydrate chains (Clarke and Shannon, 1976) and possibly one or two calcium ions. Although there is no X-ray structure available, there is a model, based on the sequence homology of HRP to the cytochrome-c peroxidase (Welinder, 1985).The catalytic mechanism of HRP has been well studied and the intermediate states (compounds 0, I and 11) characterised (Jones and Dunford, 1977;Adediran and Dunford, 1983; Van Waart, 1989, 1992;Ator and Ortiz de Montellano, 1987 A detailed analysis of the structure and function of H W would be aided by introducing point mutations and studying their effects on the catalytic activity and binding affinity of the substrates. Successful experiments with other haemoproteins expressed in Escherichia coli (Fishel et al., 1987, Phillips et al., 1990) encouraged us to express the synthetic gene for HRP, developed by Ortlepp et al. (1989), in E. coli. The E. coli system would not only facilitate mutagenesis work but would also produce non-glycosylated recombinant HRP, providing a possible starting material for X-ray crystallograAlthough HRP w...

show abstract

Section: Stabilising Effect Of Ca2+ On the Hrpmentioning

confidence: 99%

A step towards understanding the folding mechanism of horseradish peroxidase

Pappa

Cass

1993

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Horseradish isoperoxidases HRP A and HRP C have been analyzed for Ca2+ content by Haschke and Friedhoff [37], HRP BI, HRP B2, HRP B3, HRP C1 and HRP C2 by Aibara et al [38] by atomic absorption spectrometry. All contain two Ca2+ per molecule.…”

Section: + In Plant Peroxidasesmentioning

confidence: 99%

“…All contain two Ca2+ per molecule. Ca2' has never been related to reaction mechanisms, but Haschke and Friedhoff [37] suggested a role in structure stability, as removal of Ca2+ from HRP C decreased the specific activity moderately, and the thermal stability drastically. The Ca2' content of turnip peroxidase TP 7 and of yeast cytochrome c peroxidase has not been analyzed similarly.…”

Section: + In Plant Peroxidasesmentioning

confidence: 99%

Plant peroxidases. Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase

Welinder

1985

Eur J Biochem

165

View full text Add to dashboard Cite

The amino acid sequences of the 51% different horseradish peroxidase HRP C and turnip peroxidase TP 7 have previously been completed by us, but the three-dimensional structures are unknown. Recently the amino acid sequence and the crystal structure of yeast cytochrome c peroxidase have appeared. The three known apoperoxidases consist of 300 & 8 amino acid residues. The sequences have now been aligned and show 18% and 16% identity only, between the yeast peroxidase and plant peroxidase HRP C and TP 7, respectively. We show that different structural tests all support similar protein folds in plant peroxidases and yeast peroxidase and, therefore, a common evolutionary origin. The following tests support this thesis: (a) predicted helices in the plant peroxidases follow the complex pattern observed in the crystal structure of cytochrome c peroxidase; (b) their hydropathic profiles are similar and agree with observed buried and exposed peptide chain in cytochrome c peroxidase ; (c) half-cystines which are distant in the amino acid sequence of plant peroxidases become spatial neighbours when fitted into the cytochrome c peroxidase model; (d) the two-domain structure proposed from limited proteolysis of apoperoxidase HRP C is observed in the crystal structure of cytochrome c peroxidase.The similarities and differences of the plant and yeast peroxidases and the reactive side chains of a plant peroxidase active site are described. The characteristics of Ca2+-binding sequences, derived from several superfamilies, are applied to predict the Ca2 +-binding sequences in plant peroxidases.Plant peroxidases, their derivatives and catalytic intermediates have been the subject of a vast number of kinetic and spectroscopic studies. Such studies require a detailed spatial model for full interpretation. X-Ray crystallographic analysis of a plant peroxidase, however, has so far been unsuccessful, but the primary structures of horseradish peroxidase HRP C 11, 21, turnip peroxidase TP 7 [3, 41, and of the heme-linked sequences of turnip peroxidase TP 1, TP 2 and TP 3 [5] are known. This paper explores the available amino acid sequences of plant peroxidases to predict and describe molecular and structural characteristics of plant peroxidases.Recent comparison of structural and functional properties of plant peroxidases and the known hemoprotein superfamilies pointed to cytochrome c peroxidase from yeast as a possible analog. As part of the present paper, we support the thesis that plant peroxidases and cytochrome c peroxidase of yeast are structural homologues, although the reductant of cytochrome c peroxidase is a macromolecule, whereas it is a small molecule in the case of plant peroxidases. A crude model of plant peroxidases based on the recent crystal structure of cytochrome c peroxidase solved by T. Poulos et al. [6] is proposed and discussed. It is argued that the kinetic and spectral data on plant peroxidases may now confidently be interpreted in molecular terms based on the active site geometry of cytochrome c peroxidase and t...

show abstract

“…Atomic absorption spectroscopy indicates the presence of 2 mol Caz+/mol CPRx (Hu et a]., 1987), a result similar to that found for class I1 peroxidase from the basidiomycete Phanerochuete chrysosporium (LIP; Poulos et al, 1993) and class I11 peroxidase HRPc (Haschke and Friedhoff, 1978). Removal of the Ca2+ from either CPRx or HRPc reduces their thermal stabilities Haschke and Friedhoff, 1978) and significantly decreases the specific activity of both enzymes (Rodriguez Maraii6n et al, 1993 ;Haschke and Friedhoff, 1978). Magnetic circular dichroism (MCD) measurements (Rodriguez Maraii6n et al, 1994) and kinetic measurements (Lambier et al, 1985) suggest that the effect of calcium on the heme environments of both peroxidases is very similar.…”

mentioning

confidence: 54%

“…The protein consists of a single polypeptide chain (40 kDa), three glycan chains (Wan and van Huystee, 1993) and an iron protoporphyrin IX prosthetic group (Chibbar et al, 1984). Atomic absorption spectroscopy indicates the presence of 2 mol Caz+/mol CPRx (Hu et a]., 1987), a result similar to that found for class I1 peroxidase from the basidiomycete Phanerochuete chrysosporium (LIP; Poulos et al, 1993) and class I11 peroxidase HRPc (Haschke and Friedhoff, 1978). Removal of the Ca2+ from either CPRx or HRPc reduces their thermal stabilities Haschke and Friedhoff, 1978) and significantly decreases the specific activity of both enzymes (Rodriguez Maraii6n et al, 1993 ;Haschke and Friedhoff, 1978).…”

mentioning

confidence: 65%

Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by ¹H‐NMR Spectroscopy

Barber

Marañón

Shaw

et al. 1995

European Journal of Biochemistry

View full text Add to dashboard Cite

The cationic isozyme of peanut peroxidase (CPRx) is one of many peroxidases which requires calcium for enzyme activity. It has been previously shown that it requires 2 mol calcium to coordinate to 1 mol CPRx, and its related peroxidases from the basidiomycete Phanerochaete chrysosporium (Lip) and isozyme C of horseradish (HRPc). X-ray crystallographic studies of Lip have shown that calcium is ligated near the C-terminus of helices proximal and distal to the heme, where it has been suggested to maintain the active site. To determine if such a mechanism was possible in CPRx, high resolution 'H-NMR spectroscopy was used to study the effect of calcium on the environment of its heme group and the coordinating histidine residues. The low-spin cyano complex of the enzyme (CPRxCN) was studied in order to assign the majority of the resonances arising from the protons in the heme pocket in both the presence and absence of bound calcium ions using two dimensional nuclear Overhauser effect spectroscopy (NOESY). The two calcium ions present in CPRxCN were removed by a non-denaturing method and a calcium titration was performed and monitored by 'H-NMR spectroscopy. These studies showed that the binding of both calcium ions in CPRx influenced the heme environment in a similar manner (Kd = 0.1 pM). In particular, calcium-dependent changes in several heme resonances and the proximal and distal histidine residues suggest that calcium binding to CPRx causes some reorientation of these residues with respect to the active site.

show abstract

Calcium-related properties of horseradish peroxidase

Cited by 136 publications

References 3 publications

A step towards understanding the folding mechanism of horseradish peroxidase

A step towards understanding the folding mechanism of horseradish peroxidase

Plant peroxidases. Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase

Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by ¹H‐NMR Spectroscopy

Contact Info

Product

Resources

About

Calcium-related properties of horseradish peroxidase

Cited by 136 publications

References 3 publications

A step towards understanding the folding mechanism of horseradish peroxidase

A step towards understanding the folding mechanism of horseradish peroxidase

Plant peroxidases. Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase

Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by 1H‐NMR Spectroscopy

Contact Info

Product

Resources

About

Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by ¹H‐NMR Spectroscopy