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, Asp268(94), His261(95), Glu408(242) and Met409(243), in catalytic and spectral properties of human myeloperoxidase was provided by the analysis of site-directed mutants wherein these amino acids have been substituted by asparagine, alanine, glutamine and glutamine respectively. Although none of the mutations prevented folding, heme incorporation or secretion of the enzyme from transfected Chinese Hamster Ovary cell lines, 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(95)->Ala and Met409(243)->Gln mutants totally lost the typical peroxidasic activity of the enzyme, whereas the Asp268(94)->Asn mutant was only partially active. These results confirm that His261(95) is the distal histidine essential for the catalytic activity of the enzyme while Asp268(94), Met409(243) and G1u488(242) are necessary for maintaining the correct conformation of the active site and all four residues that interact closely with the periphery of the heme contribute to the unique spectral properties of the heme in MPO. (C) 1994 Academic Press, Inc.