Inactivation of lignin peroxidase by phenylhydrazine and sodium azide.
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 delta-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 of LiP resembles the closed structure of horseradish peroxidase more than it does the open structure of the globins, catalase, chloroperoxidase, or cytochrome P450.
DePillis
G D
GD
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
Wariishi
H
H
Gold
M H
MH
Ortiz de Montellano
P R
PR
D001386:Azides / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D007700:Kinetics; D010544:Peroxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010659:Phenylhydrazines / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D019810:Sodium Azide; D013053:Spectrophotometry
D001386:Azides; D007527:Isoenzymes; D010659:Phenylhydrazines; C030299:phenylhydrazine; D019810:Sodium Azide; D006861:Hydrogen Peroxide; D010544:Peroxidases; C042858:lignin peroxidase