The role of Ala134 in controlling substrate binding and reactivity in ascorbate peroxidase

Abstract

Ascorbate peroxidase (APX) is a class I heme peroxidase. It has two sites for binding of substrates. One is close to the γ-heme edge and is used for oxidation of ascorbate; the other is at the δ-heme edge and is used for binding of aromatic substrates [Gumiero et al., (2010) Arch. Biochem. Biophys. 500, 13-20]. In this work, we have examined the structural factors that control binding at the δ-heme edge by replacement of Ala134 in APX with a proline residue that is more commonly found in other class II and III peroxidases. Kinetic data indicate that replacement of Ala134 by proline has only a small effect on the catalytic mechanism, or the oxidation of ascorbate or guaiacol. Chemical modification with phenylhydrazine indicates that heme accessibility close to the δ-heme edge is only minorly affected by the substitution. We conclude that the A134P mutation alone is not enough to substantially affect the reactivity of APX towards aromatic substrates bound at the δ-heme edge. The data are relevant to the recent application of APX (APEX) in cellular imaging.

Protein Phosphatase (PP2C9) Induces Protein Expression Differentially to Mediate Nitrogen Utilization Efficiency in Rice under Nitrogen-Deficient Condition

“…Yang et al [ 86 ] reported an upregulation of HSP 70 in Phaeodactylum tricornutum under N stress. Ascorbate peroxidase (APX) exists in isoforms in different subcellular organelles such as peroxisome, chloroplasts, cytosol, and mitochondria and helps plants detoxify H 2 O 2 by converting it into H 2 O [ 87 , 88 ]. Chaperonin 60 kDa protein (Spot 29), was upregulated in PP2C9TL under low N, these chaperonins which help and guide cells in the correct folding of proteins both in normal conditions [ 89 ] and under stress [ 90 ].…”

Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

“…the corresponding position that may promote a higher tolerance against inactivation by H 2 O 2 and/or enhancing oxidation of phenolic substrates (110,111). Yet, despite the improvements made to the enzyme, APEX2 remains less active than HRP and more sensitive to H 2 O 2 mediated inhibition (110), suggesting that further improvements may be possible.…”

Plant Proteomic Research 4.0

“…Last but not least, proteomics has been employed to analyze the proteome of seeds and changes associated to seed maturation and germination in an attempt to characterize and differentiate, at the molecular level, orthodox and non-orthodox species and zygotic and somatic embryos ( [18,19,[86][87][88][89][90][91][92][93]; this study is of great importance for propagation and seed conservation programs.…”

“…Terpenes constitute a large class of secondary metabolites that serve multiple roles in the interactions between plants and their environment, including biotic and abiotic stress responses [88]. They are involved in environmental stimuli perception, stress, and phytohormone signaling [89,90].…”

“…Yang et al [86] reported an upregulation of HSP 70 in Phaeodactylum tricornutum under N stress. Ascorbate peroxidase (APX) exists in isoforms in different subcellular organelles such as peroxisome, chloroplasts, cytosol, and mitochondria and helps plants detoxify H 2 O 2 by converting it into H 2 O [87,88]. Chaperonin 60 kDa protein (Spot 29), was upregulated in PP2C9TL under low N, these chaperonins which help and guide cells in the correct folding of proteins both in normal conditions [89] and under stress [90].…”