Structural and conformational stability of horseradish peroxidase: effect of temperature and pH.


Abstract

Detailed circular dichroism and fluorescence studies at different pHs have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme c (HRPc). The change in CD in the 222 nm region corresponds to changes in the overall secondary structure of the enzyme, while that in the 400 nm region (Soret region) corresponds to changes in the tertiary structure around the heme in the enzyme. The temperature dependence of the tertiary structure around the heme also affected the intrinsic tryptophan fluorescence emission spectrum of the enzyme. The results suggested that melting of the tertiary structure to a pre-molten globule form takes place at 45 degrees C, which is much lower than the temperature (T(m) = 74 degrees C) at which depletion of heme from the heme cavity takes place. The melting of the tertiary structure was found to be associated with a pK(a) of approximately 5, indicating that this phase possibly involves breaking of the hydrogen-bonding network of the heme pocket, keeping the heme moiety still inside it. The stability of the secondary structure of the enzyme was also found to decrease at pH below 4.5. A 'high temperature' unfolding phase was observed which was, however, independent of pH. The stability of the secondary structure was found to drastically decrease in the presence of DTT (dithiothreitol), indicating that the 'high temperature' form is possibly stabilized due to interhelical disulfide bonds. Depletion of Ca(2+) ions resulted in a marked decrease in the stability of the secondary structure of the enzyme. Study holds ProTherm entries: 7900 Extra Details: DTT (30 mM) was added in the experiment secondary structure; tertiary structure; heme cavity;,interhelical disulfide bonds

Submission Details

ID: SzZ48oWi3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Chattopadhyay K;Mazumdar S,Biochemistry (2000) Structural and conformational stability of horseradish peroxidase: effect of temperature and pH. PMID:10625502
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1GWU 2003-03-28 1.31 RECOMBINANT HORSERADISH PEROXIDASE C1A ALA140GLY
7ATJ 2000-01-14 1.47 RECOMBINANT HORSERADISH PEROXIDASE C1A COMPLEX WITH CYANIDE AND FERULIC ACID
1W4W 2005-01-19 1.55 Ferric horseradish peroxidase C1A in complex with formate
1HCH 2002-07-19 1.57 Structure of horseradish peroxidase C1A compound I
1H5L 2002-06-21 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (89-100% dose)
1W4Y 2005-01-19 1.6 Ferrous horseradish peroxidase C1A in complex with carbon monoxide
1H5H 2002-06-21 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (44-56% dose)
1H5A 2002-06-18 1.6 STRUCTURE OF FERRIC HORSERADISH PEROXIDASE C1A IN COMPLEX WITH ACETATE
1H5E 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (11-22% dose)
1H57 2002-06-17 1.6 Structure of horseradish peroxidase C1A compound III
1H5I 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (56-67% dose)
1H5K 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (78-89% dose)
1H5M 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (0-100% dose)
1H5D 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (0-11% dose)
1H5F 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (22-33% dose)
1H5G 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (33-44% dose)
1H5J 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (67-78% dose)
1H55 2002-06-18 1.61 STRUCTURE OF HORSERADISH PEROXIDASE C1A COMPOUND II
1H5C 2002-06-18 1.62 X-ray induced reduction of horseradish peroxidase C1A Compound III (100-200% dose)
2YLJ 2012-06-13 1.69 Horse Radish Peroxidase, mutant S167Y
1H58 2002-06-18 1.7 STRUCTURE OF FERROUS HORSERADISH PEROXIDASE C1A
1GWT 2003-03-28 1.7 RECOMBINANT HORSERADISH PEROXIDASE C1A PHE221MET
1KZM 2002-10-09 2.0 Distal Heme Pocket Mutant (R38S/H42E) of Recombinant Horseradish Peroxidase C (HRP C).
6ATJ 2000-01-14 2.0 RECOMBINANT HORSERADISH PEROXIDASE C COMPLEX WITH FERULIC ACID
2ATJ 1998-01-28 2.0 RECOMBINANT HORSERADISH PEROXIDASE COMPLEX WITH BENZHYDROXAMIC ACID
1GWO 2003-03-28 2.07 Recombinant horseradish peroxidase C1A ALA170GLN
1ATJ 1998-02-04 2.15 RECOMBINANT HORSERADISH PEROXIDASE C1A
1GW2 2003-03-28 2.15 RECOMBINANT HORSERADISH PEROXIDASE C1A THR171SER IN COMPLEX WITH FERULIC ACID
1GX2 2003-03-28 2.2 Recombinant horseradish peroxidase Phe209Ser complex with benzhydroxamic acid
3ATJ 1999-04-28 2.2 HEME LIGAND MUTANT OF RECOMBINANT HORSERADISH PEROXIDASE IN COMPLEX WITH BENZHYDROXAMIC ACID
4ATJ 2002-10-02 2.5 DISTAL HEME POCKET MUTANT (H42E) OF RECOMBINANT HORSERADISH PEROXIDASE IN COMPLEX WITH BENZHYDROXAMIC ACID

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Peroxidase C2 P17179 PER2_ARMRU
90.8 Peroxidase C1A P15232 PER1B_ARMRU
91.2 Peroxidase C1A P15233 PER1C_ARMRU
91.2 Peroxidase C1A P24101 PER33_ARATH
93.1 Peroxidase C1A Q9SMU8 PER34_ARATH
100.0 Peroxidase C1A P00433 PER1A_ARMRU