SITE-DIRECTED MUTAGENESIS OF HUMAN MYELOPEROXIDASE - FURTHER IDENTIFICATION OF RESIDUES INVOLVED IN CATALYTIC ACTIVITY AND HEME INTERACTION

Evidence for the involvement of four spatially clustered residues, Asp 268(94), His261(95), Glu408(242) and Met409(243), in catalytic and spe ctral properties of human myeloperoxidase was provided by the analysis of site-directed mutants wherein these amino acids have been substitu ted by asparagine, alanine, glutamine and glutamine respectively. Alth ough none of the mutations prevented folding, heme incorporation or se cretion of the enzyme from transfected Chinese Hamster Ovary cell line s, the G1u488(242) to Gin and the Met409(243) to Gin substitutions led to a full blue-shift of the Sorer peak, whereas the Asp268(94) to Asn modification led to a partial blue-shift. On the other hand, His261(9 5)->Ala and Met409(243)->Gln mutants totally lost the typical peroxida sic activity of the enzyme, whereas the Asp268(94)->Asn mutant was onl y partially active. These results confirm that His261(95) is the dista l histidine essential for the catalytic activity of the enzyme while A sp268(94), Met409(243) and G1u488(242) are necessary for maintaining t he correct conformation of the active site and all four residues that interact closely with the periphery of the heme contribute to the uniq ue spectral properties of the heme in MPO. (C) 1994 Academic Press, In c.