Elementary steps in the formation of horseradish peroxidase compound I: direct observation of compound 0, a new intermediate with a hyperporphyrin spectrum.
The reaction of horseradish peroxidase (HRP) with H2O2 has been studied in 50% v/v methanol/water over the 25.0 to -36.0 degrees C temperature range by using the low-temperature stopped-flow technique. All reactions were carried out under pseudo-first-order conditions with [H2O2] much greater than [HRP]. Arrhenius plots for the pseudo-first-order rate constant kobs were linear over the 17.6 to -36.0 degrees C temperature range studied with an activation energy of 4.8 +/- 0.5 kcal/mol. Above 0 degrees C, kobs varies linearly with peroxide concentration. However, saturation kinetics are observed below -16.0 degrees C, indicating that there is at least one reversible elementary step in this reaction. Double-reciprocal plots at -26.0 degrees C at pH* 7.3 for the reaction give kappa max(obs) = 163 s-1 and KM = 0.190 mM. Rapid-scan optical studies carried out at -35.0 degrees C with [H2O2] much greater than KM reveal the presence of a transient intermediate referred to as compound 0 whose conversion to compound I is rate limiting. The Soret region of the optical spectrum of compound 0 resembles that of a "hyperporphyrin" with prominent bands near 330 and 410 nm. The temperature dependencies of kappa max(obs) and KM have been measured over the -16.0 to -26.0 degrees C range and give an activation energy for kappa max(obs) of 1.6 +/- 0.7 kcal/mol and an enthalpy of formation for compound 0 of 4.0 +/- 0.7 kcal/mol.
Baek
H K
HK
Department of Chemistry, Florida State University, Tallahassee 32306.
Van Wart
H E
HE
D006735:Horseradish Peroxidase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D010544:Peroxidases / Q000378:metabolism*; D011166:Porphyrins / Q000378:metabolism; D013816:Thermodynamics
D011166:Porphyrins; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010544:Peroxidases