Effect of Calcium, Other Ions, and pH on the Reactions of Barley Peroxidase with Hydrogen Peroxide and Fluoride

Abstract

Transient-state kinetic analysis of compound I formation for barley grain peroxidase (BP 1) has revealed properties that are highly unusual for a heme peroxidase but which may be relevant to its biological function. The enzyme shows very little reaction with H 2 O 2 at pH > 5 and exhibited saturation kinetics at higher H 2 O 2 concentrations (k cat app increases from 1.1 s ؊1 at pH 4.5 to 4.5 s ؊1 at pH 3.1 with an enzyme-linked pK a < 3.7 (Rasmussen, C. B., Bakovic, M., Welinder, K. G., and Dunford, H. B. (1993) FEBS Lett. 321, 102-105)). In the present paper, it is shown that the presence of Ca 2؉ gives rise to biphasic kinetics for compound I formation, with a slow phase as described above and a fast phase that exhibits a second order rate constant more typical of a classical peroxidase (k 1 app ‫؍‬ 1.5 ؋ Homologous heme-containing peroxidases are found in plants, fungi, and bacteria. Plants use these enzymes in intracellular peroxide scavenging and in extracellular protective processes and hormonal signaling. Plants encode a small family (an estimated 10 genes) of soluble and membrane-bound ascorbate peroxidases found in chloroplast, cytosol, and microbodies (1-3) and a large family (more than 50 genes) of peroxidases targeted for the secretory pathway (4). Interestingly, ascorbate peroxidases have a prokaryotic origin (class I), since they are more closely related in structure to mitochondrial (exemplified by yeast cytochrome c peroxidase; CCP) 1 and bacterial peroxidases than to the secretory peroxidases of plants (class III) or of fungi (class II) (5).Horseradish peroxidase (HRP C) is the most studied and applied class III peroxidase and catalyzes the oxidation of a broad range of organic and inorganic substrates (AH) by peroxide (ROOH) via the following three-step mechanism, which applies to the majority of heme peroxidases (6).The intermediates cpd I and II are very potent oxidants and have redox potentials near 900 mV. Products are often radicals (A ⅐ ) that may continue in nonenzymatic reactions depending on their chemical reactivity and the environment.We wanted to understand how plants use and regulate the activity of such a great number of rather unspecific peroxidase enzymes and chose to study the structure-function relationships of barley grain peroxidase (BP 1), since it showed only 1% of the specific activity of HRP C under standard assay conditions at pH 5.0 (7). First, a transient-state kinetics study revealed that BP 1 cpd I formation has a pH optimum near 3.4 and that this reaction is rather slow and increases in rate as the pH is decreased, saturating with increasing hydrogen peroxide concentration, in complete contrast to HRP C (8). Second, a steady-state kinetic study at pH 4.0 with ferulic acid, caffeic acid, and coniferyl alcohol was interpreted assuming that the rate constant k 1 of BP 1 cpd I formation was enhanced in the presence of reducing substrate, although the presence of 1 mM CaCl 2 also contributed (3-fold in the case of caffeic acid) (9). In

Reversible alkaline inactivation of lignin peroxidase involves the release of both the distal and proximal site calcium ions and bishistidine co-ordination of the haem

“…Recent studies with barley peroxidase have suggested that the activity of plant peroxidases may be reversibly controlled by pH and Ca# + -induced conformation changes [27]. Our data show how isotope labelling with %&Ca#+ has potential for kinetic studies of protein folding, such as those with C. cinereus peroxidase [28], which are relevant to the solubilization from inclusion bodies and refolding of variant recombinant peroxidases for academic research and commercial purposes [29,30].…”

Structure of Barley Grain Peroxidase Refined at 1.9-Å Resolution

“…Active Site-Kinetic analysis of BP 1 suggests that BP 1 has a distal histidine in the common arrangement with heme after pH-induced activation (3). The activation has a pK a Ͻ 3.7, and the resultant peroxidase exhibits slow compound I formation with unusual kinetic properties.…”

“…The possibility of an alternative site of calcium binding with K D ϭ 4 mM leading to a BP 1 with typical peroxidase kinetics is discussed in Ref. 3.…”

“…Still, with its unique pH and calcium dependent activity profile (2,3), BP 1 is the most interesting and atypical of the class III peroxidases. In the presence of calcium and following a pHinduced conformational change with a pK a Ͻ 3.7, BP 1 has a rate constant for compound I formation comparable with the rate constant for HRP C. The similarities between BP 1 and HRP C include N-terminal signal peptides, C-terminal propeptides suggesting a vacuolar targeting, isoelectric points near 9, four disulfide bridges, and an amino acid sequence identity of 42% including all active site residues (4).…”

A Review: Biological and Technological Functions of Barley Seed Pathogenesis-Related Proteins (PRs)

“…In common with other heme-containing peroxidases, BP1 catalyzes the following multistep reaction 271 The rate of the BP1 reaction with hydrogen peroxide is very slow at a pH above 5 and increases as the pH is lowered to 3. At acidic pH, in the presence of calcium ions, the kinetics becomes biphasic 270 and the fast phase behaves like HRPC 272 .…”