Molecular characterization of thylakoid membrane-bound ascorbate peroxidase in oryza sativa (Rice)

Abstract

A comparative kinetic analysis and enzymic inactivation in ascorbate depleted medium between thylakoid membrane-bound ascorbate peroxidase (tAPx) and cytosolic isoform of APx (cAPx1 and cAPx2) purified from rice (Oryza sativa L.) leaves, were performed. The three APxs follow the peroxidase ping-pong mechanism to catalyze the oxidation of ascorbate, with different kmASc and kmH 2 O 2values. They share similarly enzymatic properties so far, with the exception that tAPx was more inactivation in ascorbate depleted medium than cAPxs. Three dimensional structural analysis and alignment of amino acid sequence deduced from cloned cDNA show that thylakoid-bound and cytosol APxs share the same active site composed of two substrate-binding sites: The distal histidine site where H2O2 binds and the γ-heme edge site where the ascorbate binds. Noteworthyly, an additional loop structure composed of 16 amino acid residues was found only in tAPx. The rapid inactivation of tAPX might be due to the additional loop structure, which forms an access channel connecting the molecular surface of tAPX to the γ-edge of heme, named γ-channel.