title: Stimulation of the activity of horseradish peroxidase by nitrogenous compounds.
abstract: A variety of nitrogenous compounds broaden the activity versus pH profile for the peroxidation of dianisidine catalyzed by horseradish peroxidase (HRP), but not by myeloperoxidase, chloroperoxidase, Escherichia coli hydroperoxidase I, methemoglobin, or microperoxidases. The peroxidation of dianisidine catalyzed by cytochrome c peroxidase was affected by the nitrogenous compounds, but to a lesser extent than was the action of HRP. The peroxidations of a variety of phenols by HRP exhibited broad activity versus pH profiles and were unaffected by the nitrogenous compounds. The energy of activation for the peroxidation of dianisidine by HRP was unaffected by changes of pH in the range 6.5-8.5 and was unchanged by the presence of the nitrogenous compounds. The nitrogenous compounds markedly increased Vm for the peroxidation of dianisidine by HRP, but did not change the slope of Lineweaver-Burk plots of kinetic data. These results are accommodated by a mechanism in which nitrogenous compounds hydrogen-bond to the distal histidine of HRP and in so doing raise its pK alpha. Since the acid form of the distal histidine is thought to facilitate peroxidations catalyzed by HRP by hydrogen bonding to the ferryl oxygen of compound II, raising its pK alpha broadens the activity versus pH profile for the peroxidation of anilino substrates, such as dianisidine. We propose that phenolic substrates hydrogen-bond directly to the ferryl oxygen, thus displacing the distal histidine and eliminating the possibility of being influenced by nitrogenous compounds.
authors.affiliation: Department of Biochemistry, Duke University of Medical Center, Durham, North Carolina 27710.
authors.forename: C F
authors.initials: CF
authors.lastname: Kuo
authors.forename: I
authors.initials: I
authors.lastname: Fridovich
mesh_terms: D000588:Amines / Q000494:pharmacology*; D003578:Cytochrome-c Peroxidase / Q000378:metabolism; D003962:Dianisidine; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D010544:Peroxidases / Q000378:metabolism*
chemical_list: D000588:Amines; D007093:Imidazoles; C029899:imidazole; D006735:Horseradish Peroxidase; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase; D003962:Dianisidine