Inactivation of lignin peroxidase by phenylhydrazine and sodium azide

Gia D. DePillis, Hiroyuki Wariishi, Michael H. Gold, Paul R.Ortiz de Montellano

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Lignin peroxidase (LiP) is rapidly inactivated in a concentration-dependent manner by H2O2 and either phenylhydrazine or sodium azide. Full inactivation of isozyme 2b (H8) requires approximately 50 eq of phenylhydrazine or 80 eq of sodium azide. Anaerobic incubation of isozyme 2b with [14C]phenylhydrazine and H2O2 results in 77% loss of catalytic activity and covalent binding of 0.45 mol radiolabel/mol of enzyme. Comparable but not identical results are obtained with an isozyme mixture. A lag period is observed before the peroxidative activity can be measured when an aliquot of an incubation with sodium azide is diluted into the mixture used to assay residual catalytic activity. This lag is associated with reversible accumulation of a catalytically inert species with a Compound III-like spectrum. No meso-phenyl, iron-phenyl, or N-phenyl adducts are formed with phenylhydrazine but a low yield of what appears to be δ-meso-azidoheme is obtained with sodium azide. LiP is thus less susceptible to meso heme additions and more susceptible to oxidative heme degradation than horseradish peroxidase. The data suggest that the active site of LiP resembles the closed structure of horseradish peroxidase more than it does the open structure of the globins, catalase, chloroperoxidase, or cytochrome P450.

Original languageEnglish
Pages (from-to)217-223
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume280
Issue number1
DOIs
Publication statusPublished - Jul 1990

Fingerprint

Sodium Azide
Isoenzymes
Horseradish Peroxidase
Heme
Catalyst activity
Chloride Peroxidase
Globins
Catalase
Cytochrome P-450 Enzyme System
Assays
Catalytic Domain
Iron
Degradation
phenylhydrazine
lignin peroxidase
Enzymes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Inactivation of lignin peroxidase by phenylhydrazine and sodium azide. / DePillis, Gia D.; Wariishi, Hiroyuki; Gold, Michael H.; de Montellano, Paul R.Ortiz.

In: Archives of Biochemistry and Biophysics, Vol. 280, No. 1, 07.1990, p. 217-223.

Research output: Contribution to journalArticle

DePillis, Gia D. ; Wariishi, Hiroyuki ; Gold, Michael H. ; de Montellano, Paul R.Ortiz. / Inactivation of lignin peroxidase by phenylhydrazine and sodium azide. In: Archives of Biochemistry and Biophysics. 1990 ; Vol. 280, No. 1. pp. 217-223.
@article{80e4d5f1d64a4bb9aaf650e311cfb0ba,
title = "Inactivation of lignin peroxidase by phenylhydrazine and sodium azide",
abstract = "Lignin peroxidase (LiP) is rapidly inactivated in a concentration-dependent manner by H2O2 and either phenylhydrazine or sodium azide. Full inactivation of isozyme 2b (H8) requires approximately 50 eq of phenylhydrazine or 80 eq of sodium azide. Anaerobic incubation of isozyme 2b with [14C]phenylhydrazine and H2O2 results in 77{\%} loss of catalytic activity and covalent binding of 0.45 mol radiolabel/mol of enzyme. Comparable but not identical results are obtained with an isozyme mixture. A lag period is observed before the peroxidative activity can be measured when an aliquot of an incubation with sodium azide is diluted into the mixture used to assay residual catalytic activity. This lag is associated with reversible accumulation of a catalytically inert species with a Compound III-like spectrum. No meso-phenyl, iron-phenyl, or N-phenyl adducts are formed with phenylhydrazine but a low yield of what appears to be δ-meso-azidoheme is obtained with sodium azide. LiP is thus less susceptible to meso heme additions and more susceptible to oxidative heme degradation than horseradish peroxidase. The data suggest that the active site of LiP resembles the closed structure of horseradish peroxidase more than it does the open structure of the globins, catalase, chloroperoxidase, or cytochrome P450.",
author = "DePillis, {Gia D.} and Hiroyuki Wariishi and Gold, {Michael H.} and {de Montellano}, {Paul R.Ortiz}",
year = "1990",
month = "7",
doi = "10.1016/0003-9861(90)90539-B",
language = "English",
volume = "280",
pages = "217--223",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Inactivation of lignin peroxidase by phenylhydrazine and sodium azide

AU - DePillis, Gia D.

AU - Wariishi, Hiroyuki

AU - Gold, Michael H.

AU - de Montellano, Paul R.Ortiz

PY - 1990/7

Y1 - 1990/7

N2 - Lignin peroxidase (LiP) is rapidly inactivated in a concentration-dependent manner by H2O2 and either phenylhydrazine or sodium azide. Full inactivation of isozyme 2b (H8) requires approximately 50 eq of phenylhydrazine or 80 eq of sodium azide. Anaerobic incubation of isozyme 2b with [14C]phenylhydrazine and H2O2 results in 77% loss of catalytic activity and covalent binding of 0.45 mol radiolabel/mol of enzyme. Comparable but not identical results are obtained with an isozyme mixture. A lag period is observed before the peroxidative activity can be measured when an aliquot of an incubation with sodium azide is diluted into the mixture used to assay residual catalytic activity. This lag is associated with reversible accumulation of a catalytically inert species with a Compound III-like spectrum. No meso-phenyl, iron-phenyl, or N-phenyl adducts are formed with phenylhydrazine but a low yield of what appears to be δ-meso-azidoheme is obtained with sodium azide. LiP is thus less susceptible to meso heme additions and more susceptible to oxidative heme degradation than horseradish peroxidase. The data suggest that the active site of LiP resembles the closed structure of horseradish peroxidase more than it does the open structure of the globins, catalase, chloroperoxidase, or cytochrome P450.

AB - Lignin peroxidase (LiP) is rapidly inactivated in a concentration-dependent manner by H2O2 and either phenylhydrazine or sodium azide. Full inactivation of isozyme 2b (H8) requires approximately 50 eq of phenylhydrazine or 80 eq of sodium azide. Anaerobic incubation of isozyme 2b with [14C]phenylhydrazine and H2O2 results in 77% loss of catalytic activity and covalent binding of 0.45 mol radiolabel/mol of enzyme. Comparable but not identical results are obtained with an isozyme mixture. A lag period is observed before the peroxidative activity can be measured when an aliquot of an incubation with sodium azide is diluted into the mixture used to assay residual catalytic activity. This lag is associated with reversible accumulation of a catalytically inert species with a Compound III-like spectrum. No meso-phenyl, iron-phenyl, or N-phenyl adducts are formed with phenylhydrazine but a low yield of what appears to be δ-meso-azidoheme is obtained with sodium azide. LiP is thus less susceptible to meso heme additions and more susceptible to oxidative heme degradation than horseradish peroxidase. The data suggest that the active site of LiP resembles the closed structure of horseradish peroxidase more than it does the open structure of the globins, catalase, chloroperoxidase, or cytochrome P450.

UR - http://www.scopus.com/inward/record.url?scp=0025324114&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025324114&partnerID=8YFLogxK

U2 - 10.1016/0003-9861(90)90539-B

DO - 10.1016/0003-9861(90)90539-B

M3 - Article

C2 - 2353822

AN - SCOPUS:0025324114

VL - 280

SP - 217

EP - 223

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1

ER -