Biochemical characterization of selenium-containing catalytic antibody as a cytosolic glutathione peroxidase mimic

Abstract

A selenium-containing catalytic antibody (Se-4A4), prepared by converting reactive serine residues of a monoclonal antibody (4A4) raised against a GSH derivative into selenocysteines, acts as a mimic of cytosolic glutathione peroxidase (cGPX). To clarify the mechanism of action of this catalytic antibody, detailed studies on kinetic behaviour and biological activity were carried out. A rate of acceleration (kcat/Km/kuncat) 10(7)-fold that of the uncatalytic reaction is observed. Under similar conditions, the turnover number (kcat) of Se-4A4 is 42% of that of the natural rabbit liver cGPX. The Se-4A4 reaction involves a Ping Pong mechanism, which is the same as that of the natural cGPX. The selenocysteine residue is located in the binding site of the antibody and is shown to be crucial for this activity. Of the thiol compounds tested, only GSH is able to serve as substrate for Se-4A4. It was demonstrated, using the free-radical-damage system (hypoxanthine/xanthine oxidase) of cardiac mitochondria, that Se-4A4 can protect mitochondria from free-radical damage at least 10(4)-fold more effectively than the natural cGPX.

Substituting Selenocysteine for Catalytic Cysteine 41 Enhances Enzymatic Activity of Plant Phospholipid Hydroperoxide Glutathione Peroxidase Expressed in Escherichia coli

“…Other strategies were developed to overcome the constraint of engineering SE-CIS in the ORF. Sec insertion into the polypeptide structure has been achieved by chemical modification of Ser residue (22,23) or mischarging of tRNA Cys with Sec when Cys was omitted from the growth medium (24,25). However, these strategies do not afford directed substitution, because all Ser or Cys residues along the recombinant protein are subjected to Sec conversion.…”

Engineering Glutathione Transferase to a Novel Glutathione Peroxidase Mimic With High Catalytic Efficiency

“…3, and the standard deviations are shown in parentheses. (3), and other high efficiency GPx-like biocatalysts, such as selenium-containing catalytic antibody (5) and imprinted proteins (6). But non-site-directed substitution hampers further structure-function characterization of as-generated proteins.…”

“…However, the mechanism that these two selenoenzymes applied to catalyze the reduction of hydroperoxide involves use of aryl thiols instead of GSH as reducing substrate due to the lack of a GSH-specific binding site in their active sites. To enhance the GPx activities, the GSH binding site has been successfully introduced into GPx mimics using monoclonal antibody (5) and bioimprinting techniques (6), and the as-synthesized selenoantibody and bioimprinted selenoprotein containing the GSH binding site exhibited high GPx activities. Besides the efficient binding of substrates, however, some other factors, such as the correct geometric conformation and the microenvironment of the active site, are also dominant for the enhancement of enzyme catalysis.…”

Engineering human seleno-glutaredoxin containing consecutive rare codons as an artificial glutathione peroxidase

“…Based on the detailed structural and kinetic analysis of the natural GPx and its mutants, it was assumed that the high affinity of GSH toward the catalytic center and the optimum orientation of the sulfhydryl group of GSH to the catalytic selenium group would be critical for constructing a highly efficient GPx for catalyzing the reduction of H 2 O 2 by GSH [10]. A GSH binding site has been successfully introduced into GPx models using a monoclonal antibody [11] and bioimprinting techniques [12]. The resulting seleniumcontaining proteins exhibited high GPx activity.…”