Abstract
1. The kinetics of formation of horseradish peroxidase Compound I were studied by using peroxobenzoic acid and ten substituted peroxobenzoic acids as substrates. Kinetic data for the formation of Compound I with H2O2 and for the reaction of deuteroferrihaem with H2O2 and peroxobenzoic acids, to form a peroxidatically active intermediate, are included for comparison. 2. The observed second-order rate constants for the formation of Compound I with peroxobenzoic acids decrease with increasing pH, in the range pH 5-10, in contrast with pH-independence of the reaction with H2O2. The results imply that the formation of Compound I involves a reaction between the enzyme and un-ionized hydroperoxide molecules. 3. The maximal rate constants for Compound I formation with unhindered peroxobenzoic acids exceed that for H2O2. Peroxobenzoic acids with bulky ortho substituents show marked adverse steric effects. The pattern of substituent effects does not agree with expectations for an electrophilic oxidation of the enzyme by peroxoacid molecules in aqueous solution, but is in agreement with that expected for a reaction involving nucleophilic attack by peroxo anions. 4. Possible reaction mechanisms are considered by which the apparent conflict between the pH-effect and substituent-effect data may be resolved. A model in which it is postulated that a negatively charged 'electrostatic gate' controls access of substrate to the active site and may also activate substrate within the active site, provides the most satisfactory explanation for both the present results and data from the literature.
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Claims 8
Claim · model-written summary
Compound I formation rate constants with peroxobenzoic acids declined as pH rose.
- Taxon
- horseradish
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Armoracia rusticanat0 - Gene
- horseradish peroxidaset0
- Outcome
- second-order rate constants for the formation of Compound Ie0
- Direction
- decreasee0
- Qualifier
- pH 5-10e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
.jpg?width=500)
Verbatim
e0“The observed second-order rate constants for the formation of Compound I with peroxobenzoic acids decrease with increasing pH, in the range pH 5-10”
t0“horseradish peroxidase”
Claim · model-written summary
Compound I formation with H2O2 was independent of pH.
- Taxon
- horseradishArmoracia rusticanat0
- Gene
- horseradish peroxidaset0
- Outcome
- reaction with H2O2e0
- Direction
- pH-independencee0
- Qualifier
- pHe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“in contrast with pH-independence of the reaction with H2O2.”
t0“horseradish peroxidase”
Claim · model-written summary
Maximal Compound I formation rates with unhindered peroxobenzoic acids exceeded the H2O2 rate.
- Taxon
- horseradishArmoracia rusticanat0
- Gene
- horseradish peroxidaset0
- Outcome
- maximal rate constants for Compound I formatione0
- Direction
- exceede0
- Comparator
- H2O2e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The maximal rate constants for Compound I formation with unhindered peroxobenzoic acids exceed that for H2O2.”
t0“horseradish peroxidase”
Claim · model-written summary
Bulky ortho substituents of peroxobenzoic acids had adverse steric effects.
- Taxon
- horseradishArmoracia rusticanat0
- Gene
- horseradish peroxidaset0
- Outcome
- marked adverse steric effectse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Peroxobenzoic acids with bulky ortho substituents show marked adverse steric effects.”
t0“horseradish peroxidase”
Claim · model-written summary
Substituent effect patterns disagreed with an electrophilic oxidation expectation.
- Taxon
- horseradishArmoracia rusticanat0
- Gene
- horseradish peroxidaset0
- Outcome
- pattern of substituent effects does not agree with expectations for an electrophilic oxidatione0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The pattern of substituent effects does not agree with expectations for an electrophilic oxidation of the enzyme by peroxoacid molecules in aqueous solution”
t0“horseradish peroxidase”
Claim · model-written summary
Substituent effect patterns agreed with expected nucleophilic attack by peroxo anions.
- Taxon
- horseradishArmoracia rusticanat0
- Gene
- horseradish peroxidaset0
- Outcome
- agreement with that expected for a reaction involving nucleophilic attack by peroxo anionse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“but is in agreement with that expected for a reaction involving nucleophilic attack by peroxo anions.”
t0“horseradish peroxidase”
Claim · model-written summary
The authors infer Compound I forms through reaction with un-ionized hydroperoxide molecules.
- Taxon
- horseradishArmoracia rusticanat0
- Gene
- horseradish peroxidaset0
- Outcome
- formation of Compound Ie0
- Qualifier
- reaction between the enzyme and un-ionized hydroperoxide moleculese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The results imply that the formation of Compound I involves a reaction between the enzyme and un-ionized hydroperoxide molecules.”
t0“horseradish peroxidase”
Claim · model-written summary
The authors propose an electrostatic gate model to explain pH and substituent effects.
- Taxon
- horseradishArmoracia rusticanat0
- Gene
- horseradish peroxidaset0
- Outcome
- controls access of substrate to the active sitee0
- Qualifier
- negatively charged 'electrostatic gate'e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“A model in which it is postulated that a negatively charged 'electrostatic gate' controls access of substrate to the active site and may also activate substrate within the active site, provides the most satisfactory explanation for both the present results and data from the literature.”
t0“horseradish peroxidase”