Kinetic and spectral properties of pea cytosolic ascorbate peroxidase

Abstract

Sufficient highly purified native pea cytosolic ascorbate peroxidase was obtained to characterize some of its kinetic and spectral properties. Its rate constant for compound I formation from reaction with Hz02 is 4.0X 10' M-' s-l, somewhat faster than is typical for peroxidases. Compound I has the typical optical spectrum of an iron(porphyrin-n-cation radical, despite considerable homology with yeast cytochrome c peroxidase. The rate constant for cornround I reduction by ascorbate is extremely fast (8.0 X 10' M-s ' at pH 7.8), again in marked contrast to the behavior of the yeast enzyme. The pHrate profde for compound I formation indicates a pK, value of 5.0 for a group affecting the active site reaction.

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

“…For those that exist predominantly as homodimers, such as the cytosolic APXs from peas and soybean, pseudo first-order kinetics have been observed (33,34). In contrast, monomeric forms such as the peroxisomal APX from spinach and APXs from various unicellular algae obey Michaelis-Menten kinetics (35,36), which has led to the suggestion that the kinetics displayed by the homodimeric enzymes may be due to variation in the oligomeric form of the protein, especially when ascorbate concentrations are low (33).…”

Effect of Distal Cavity Mutations on the Formation of Compound I in Catalase-Peroxidases

“…Second, the mutants W122F and W122A for the first time allowed us to monitor the direct reaction of a catalase-peroxidase with hydrogen peroxide, resulting in an enzyme intermediate with spectral features also similar to the classical plant peroxidase compound I (including APX) (Fig. 3A), which has been shown to contain two oxidizing equivalents (13) and forms a porphyrin -cation radical in combination with an iron(IV) center (17,27). Since the Trp mutants completely lost their catalase activity but retained their peroxidase activity, it is reasonable to assume that Trp-122 is the crucial residue in compound I reduction by H 2 O 2 (Reaction 2 (a and b) in Fig.…”

Total Conversion of Bifunctional Catalase-Peroxidase (KatG) to Monofunctional Peroxidase by Exchange of a Conserved Distal Side Tyrosine

“…Thus, in contrast to wild-type KatG, Y249F behaves like horseradish peroxidase (12) or APX (19) and compound I reduction by a one-electron donor can be represented by Reaction 3 in Fig. 2.…”