PROBING THE PRESUMED CATALYTIC TRIAD OF A SELENIUM-CONTAINING PEROXIDASE BY MUTATIONAL ANALYSIS

Glutathione peroxidases (GPx) are characterized by a catalytically act ive selenium which forms the center of a strictly conserved triad comp osed of selenocysteine, glutamine, and tryptophan. In order to check t he functional relevance of this structural peculiarity, six molecular mutants of phospholipid hydroperoxide glutathione peroxidase (PHGPx) w ere designed, isolated, and investigated kinetically. Replacement of t he selenocysteine in position 46 by cysteine decreased k(+1) i.e., the reaction rate of reduced enzyme with hydroperoxide, by three orders o f magnitude. The rate of regeneration of the reduced enzyme by glutath ione (k'(+2)) was similarly affected. Additional substitution of Gln81 or Trp136 by acid residues resulted in a further decrease of k(+1), b y three orders of magnitude, whereas histidine or neutral residues in these positions proved to be less deleterious. The data support the hy pothesis that the typical triad of selenocysteine, glutamine, and tryp tophan is indeed a novel catalytic center in which the reactivity of s elenium is optimized by hydrogen bonding provided by the adjacent glut amine and tryptophan residues.