Mutation of residues critical for benzohydroxamic acid binding to horseradish peroxidase isoenzyme C

Biopolymers ◽  
2001 ◽  
Vol 62 (5) ◽  
pp. 261-267 ◽  
Author(s):  
Barry D. Howes ◽  
Hendrik A. Heering ◽  
Thomas O. Roberts ◽  
Florence Schneider-Belhadadd ◽  
Andrew T. Smith ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Peroxidase Isoenzyme ◽  
Benzohydroxamic Acid

scholarly journals The effect on structural and solvent water molecules of substrate binding to ferric horseradish peroxidase

2015 ◽  
Vol 177 ◽  
pp. 163-179 ◽  
Author(s):  
Niall Simpson ◽  
Katrin Adamczyk ◽  
Gordon Hithell ◽  
Daniel J. Shaw ◽  
Gregory M. Greetham ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Structural Dynamics ◽  
Solvent Accessibility ◽  
Substrate Binding ◽  
Dynamic Environment ◽  
Water Molecules ◽  
Pump Probe ◽  
Benzohydroxamic Acid ◽  
Enzymatic Mechanism ◽  
2D Ir

Ultrafast, multi-dimensional infrared spectroscopy, in the form of 2D-IR and pump–probe measurements, has been employed to investigate the effect of substrate binding on the structural dynamics of the horseradish peroxidase (HRP) enzyme. Using nitric oxide bound to the ferric haem of HRP as a sensitive probe of local dynamics, we report measurements of the frequency fluctuations (spectral diffusion) and vibrational lifetime of the NO stretching mode with benzohydroxamic acid (BHA) located in the substrate-binding position at the periphery of the haem pocket, in both D2O and H2O solvents. The results reveal that, with BHA bound to the enzyme, the local structural dynamics are insensitive to H/D exchange. These results are in stark contrast to those found in studies of the substrate-free enzyme, which demonstrated that the local chemical and dynamic environment of the haem ligand is influenced by water molecules. In light of the large changes in solvent accessibility caused by substrate binding, we discuss the potential for varying roles for the solvent in the haem pocket of HRP at different stages along the reaction coordinate of the enzymatic mechanism.

Preparation of aqueous polyaniline-vesicle suspensions with class III peroxidases. Comparison between horseradish peroxidase isoenzyme C and soybean peroxidase

2013 ◽  
Vol 67 (8) ◽  
Author(s):  
Katja Junker ◽  
Ivan Gitsov ◽  
Nick Quade ◽  
Peter Walde
Keyword(s):  
Horseradish Peroxidase ◽  
Enzyme Stability ◽  
Class Iii ◽  
Salt Form ◽  
Reaction Conditions ◽  
Soybean Peroxidase ◽  
Peroxidase Isoenzyme ◽  
Complete Inactivation ◽  
Class Iii Peroxidases ◽  
Optimal Reaction

AbstractAniline was polymerised enzymatically in aqueous solution at pH = 4.3 and 25°C in the presence of submicrometer-sized vesicles formed from sodium bis(2-ethylhexyl)sulphosuccinate (AOT). H2O2 served as oxidant and the enzyme used was either horseradish peroxidase isoenzyme C (HRPC) or soybean peroxidase (SBP), both being class III peroxidases. From previous studies with HRPC, it is known that stable vesicle suspensions containing the emeraldine salt form of polyaniline (PANI-ES) can be obtained within 1–2 days with a 90–95 % yield, provided that optimal reaction conditions are applied. Unfortunately, HRPC becomes inactivated during polymerisation. In the present study, a linear dendritic block copolymer was added to HRPC, resulting in higher operational enzyme stability; the stabilising effect, however, was too small to afford a substantial decrease in the required amount of enzyme. Moreover, replacing HRPC with SBP was of no advantage, although SBP is known to be more stable towards inactivation by H2O2 than HRPC. By contrast, SBP was found to be much slower in oxidising aniline, and complete inactivation of SBP occurred before all the aniline monomers were oxidised, leading to low yields and the formation of over-oxidised products. The same was observed for HRP isoenzyme A2. Reactions without vesicles indicated that peroxidase inactivation was probably caused by PANI-ES.

scholarly journals Mechanism of horseradish peroxidase inactivation by benzhydrazide: a critical evaluation of arylhydrazides as peroxidase inhibitors

2003 ◽  
Vol 375 (3) ◽  
pp. 613-621 ◽  
Author(s):  
Susan M. AITKEN ◽  
Marc OUELLET ◽  
M. David PERCIVAL ◽  
Ann M. ENGLISH
Keyword(s):  
Horseradish Peroxidase ◽  
Cytochromes P450 ◽  
Critical Evaluation ◽  
Binding Affinities ◽  
Peroxidase Isoenzyme ◽  
Inactivation Rate Constant ◽  
Radical Production ◽  
Catalytic Intermediates ◽  
Ic50 Values ◽  
Benzoyl Radical

Many compounds are oxidized by haem enzymes, such as peroxidases and cytochromes P450, to highly reactive intermediates that function as enzyme inactivators. To evaluate the potential of arylhydrazides as selective metabolically activated peroxidase inhibitors, the mechanism of HRPC (horseradish peroxidase isoenzyme C) inhibition by BZH (benzhydrazide) was investigated in detail. No oxygen consumption was detected in BZH solutions at pH 7.0–12.0, but addition of HRPC resulted in significant O2 uptake above pH 8.0, indicating that the enzyme catalyses BZH oxidation. Addition of H2O2 to HRPC plus BZH activates the latter as an inhibitor. This involves the three-electron oxidation of BZH in one-electron steps by the peroxidase catalytic intermediates, Compounds I and II, to produce a benzoyl radical that covalently alters the active site and inhibits peroxidase activity. Alternatively, the benzoyl radical could be produced by di-imide (NH=NH) elimination from the BZH radical. Production of Compound III (oxyperoxidase) followed by p-670 (m/z=583, biliverdin-like derivative) was observed for HRPC incubated with excess H2O2, and the addition of BZH resulted in an increase in the rate of p-670 production. BZH is an inefficient inhibitor of HRPC with a KI of 80 μM, an apparent inactivation rate constant (kinact) of 0.035 min−1, and an IC50 of 1.0 mM. This prompted the investigation of HRPC inactivation by a series of related arylhydrazides with known binding affinities for HRPC. The hydrazide with the highest affinity (2-naphthoichydrazide; Kd=5.2 μM) was also found to be the most effective inhibitor with KI, kinact and IC50 values of 14 μM, 0.14 min−1 and 35 μM, respectively.

The inactivation of horseradish peroxidase isoenzyme AZ by hydrogen peroxide: an example of partial resistance due to the formation of a stable enzyme intermediate

2001 ◽  
Vol 6 (5-6) ◽  
pp. 504-516 ◽  
Author(s):  
Alexander N. P. Hiner ◽  
Josefa Hernández-Ruiz ◽  
José Neptuno Rodríguez-López ◽  
Marino B. Arnao ◽  
Ramón Varón ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Horseradish Peroxidase ◽  
Partial Resistance ◽  
Peroxidase Isoenzyme ◽  
Enzyme Intermediate

Spectroscopic characterization of mutations at the Phe41 position in the distal haem pocket of horseradish peroxidase C: structural and functional consequences

2002 ◽  
Vol 363 (3) ◽  
pp. 571-579 ◽  
Author(s):  
Hendrik A. HEERING ◽  
Andrew T. SMITH ◽  
Giulietta SMULEVICH
Keyword(s):  
Horseradish Peroxidase ◽  
Fold Increase ◽  
Spectroscopic Characterization ◽  
Wild Type ◽  
Compound I ◽  
Peroxidase Isoenzyme ◽  
Mutant Proteins ◽  
Aromatic Donor ◽  
Form Compound ◽  
Trp Mutant

Three mutants of horseradish peroxidase isoenzyme C (HRPC) have been constructed in which the conserved distal aromatic residue Phe41 has been substituted by Trp, Val or Ala and the properties of the mutant proteins have been compared with that of the wild-type. The ferric and ferrous states have been studied by resonance Raman, electronic absorption and Fourier-transform infrared spectroscopies, together with their respective fluoride and CO complexes as probes for the integrity of the distal haem-pocket hydrogen-bonding network. The catalytic properties of the mutants, most notably the HRPC-mutant Phe41→Trp (F41W) variant, were also affected. Structural modelling suggests that the bulky indole group of the F41W mutant blocks the distal cavity, inhibiting the binding of fluoride and CO to the haem iron, severely impairing the reaction of the enzyme with H2O2 to form Compound I. Substitution with the smaller side-chain residues Val or Ala resulted in a 2-fold increase in the affinity of the mutants for the aromatic donor benzhydroxamic acid (BHA) compared with the wild-type, whereas the sterically hindered F41W mutant was not able to bind BHA at all. All the mutations studied increased the amount of a ferric six-coordinate aquo-high-spin species. On the other hand, the similarity in the Fe—Im stretching frequencies of the mutants and wild-type protein suggests that the distal haem-pocket mutations do not cause any substantive changes on the proximal side of the haem. Spectra of the HRPC mutant Phe41→Ala—CO and the HRPC mutant Phe41→Val—CO complexes strongly suggested a weakening of the interaction between CO and Arg38 due to a secondary rearrangement of the haem relative to helix B. The effects observed for these HRP mutants were somewhat different from those noted recently for the analogous Coprinus cinereus peroxidase (CIP) mutants, particularly the Trp mutant. These differences can be reconciled in part as being due to the smaller size of the distal cavity of HRP compared with that of CIP.

scholarly journals Effect of template type on the preparation of the emeraldine salt form of polyaniline (PANI-ES) with horseradish peroxidase isoenzyme C (HRPC) and hydrogen peroxide

RSC Advances ◽  
2019 ◽  
Vol 9 (57) ◽  
pp. 33080-33095 ◽  
Author(s):  
Tomoyuki Fujisaki ◽  
Keita Kashima ◽  
Sandra Serrano-Luginbühl ◽  
Reinhard Kissner ◽  
Danica Bajuk-Bogdanović ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Horseradish Peroxidase ◽  
Emeraldine Salt ◽  
Salt Form ◽  
Peroxidase Isoenzyme ◽  
Different Types

Different types of templates consisting of sulfonate or sulfate groups were compared for the horseradish peroxidase/H2O2-catalysed synthesis of the emeraldine salt form of polyaniline from aniline at pH = 4.3.

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