PROBING THE PRESUMED CATALYTIC TRIAD OF SELENIUM-CONTAINING PEROXIDASES BY MUTATIONAL ANALYSIS OF PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE-PEROXIDASE (PHGPX)

Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx were subjected to functional analysis, The rate constants k(+1) and k'(+2), for the oxidation and the regeneratio n of the ground state enzyme were estimated, respectively, Moreover, t he alkylation rate of the reactive centre by iodoacetate (k(inact)) wa s also analysed, The substitution of the catalytically competent selen ocysteine 46 by cysteine (PHGPx(cys46)) decreased k(+1) and k'(+2) by about three orders of magnitude, although leaving unaffected k(inact). Furthermore, mutations of PHGPx(cys46) involving the other residues o f the triad decreased both k(inact), and k(+1), thus highlighting the involvement of Gln 81 and Trp 136 in the dissociation/activation of th e nucleophilic cysteine thiol. In general, substitutions of Gin 81 or Trp 136 by acidic residues in PHGPx(cys46) most dramatically depressed the k(+1) values, because they practically prevented the dissociation of the thiol group, while neutral or positively charged residues in t hese positions allowed and intermediate dissociation and induced a cor responding reactivity of the thiol. Our data, for the first time, reve al that the presumed triad of selenocysteine, glutamine and tryptophan residues represents a novel type of catalytic centre, whose integrity is essential for the full catalytic function of glutathione peroxidas es.