Kinetic and thermodynamic behaviour of wall-bound peroxidase from wheat leaves infected with stripe rust |
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Authors: | Bavita Asthir Anjali Koundal Navtej Singh Bains |
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Affiliation: | (1) Department of Biochemistry, Punjab Agricultural University, Ludhiana, Punjab, 141004, India;(2) Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, Punjab, 141004, India |
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Abstract: | We have identified two types of peroxidases (POX), one ionically and one covalently bound to the particulate fraction, in stripe rust-infected and -uninfected wheat (Triticum aestivum L.) leaves. The cell walls contained a high level of POX, of which 73–76% was extractable by 1% NaCl and 24–26% by 5 mM EDTA in infected and non-infected leaves of HD 2329. The NaCl-released POX constituted the predominant fraction. Both NaCl- and EDTA-extracted POX exhibited maximum activity at pH 5.0 and had a K m (enzyme–substrate affinity measure) value of 1.61–1.70 and 1.64–1.67 mM, respectively, with o-dianisidine as the substrate. The V max (maximum catalytic rate) in the two extractions ranged between 7.06–7.45 and 6.65–7.82 μmol min−1 g−1 fresh weight. A temperature optimum of 50°C was observed for both the NaCl- and EDTA-released fractions. The two POX fractions showed a differential response to metal ions, suggesting their distinctive nature. Sodium azide inhibited POX activity markedly, which suggested the presence of heme as a prosthetic group. Inhibition of wall-bound POX by iodine and the regeneration of activity by mercaptoethanol suggested the involvement of cysteine in the active site of the enzyme. These two forms showed greater differences in terms of thermodynamic properties, such as the energy of activation (E a) and enthalpy change (ΔH), while entropy (ΔS) and free energy changes were similar. The results further show that pathogen infection of the leaves of this susceptible wheat cultivar induces an increase in the activity and kinetics of POX, which may be critical in the response of the plant cell to infection. |
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