Purification and Characterization of a Novel Class III Peroxidase Isoenzyme from Tea Leaves

Author:

Kvaratskhelia M.1,Winkel C.1,Thorneley RNF.1

Affiliation:

1. Nitrogen Fixation Laboratory, John Innes Centre, Norwich, United Kingdom NR4 7UH (M.K., R.N.F.T.)

Abstract

Abstract A novel, basic (isoelectric point > 10), heme peroxidase isoenzyme (TP; relative molecular weight = 34,660 [plus or minus] 10, mean [plus or minus] SE) that can account for a significant part of the ascorbate peroxidase activity in tea (Camellia sinensis) leaves has been purified to homogeneity. The ultraviolet/visible absorption spectrum is typical of heme-containing plant peroxidases, with a Soret peak at 406 nm ([epsilon] = 115 mM-1 cm-1) and an A406/A280 value of 3.4. The enzyme has a high specific activity for ascorbate oxidation (151 [mu]mol min-1 mg-1), with a pH optimum in the range of 4.5 to 5.0. Substrate-specificity studies have revealed significant differences between TP and other class III peroxidases, as well as similarities with class I ascorbate peroxidases. TP, like ascorbate peroxidase, exhibits a preference for ascorbate over guaiacol, whereas other class III isoenzymes are characterized by 2-orders-of-magnitude higher activity for guaiacol than for ascorbate. TP also forms an unstable porphyrin [pi] cation radical-type compound I, which is converted to compound II within approximately 2 min in the absence of added reductant. Amino acid sequence data show TP to be the first example, to our knowledge, of a class III peroxidase with a high specificity for ascorbate as an electron donor.

Publisher

Oxford University Press (OUP)

Subject

Plant Science,Genetics,Physiology

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