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, Asp260(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 Glu408(242) to Gln and the Met409(243) to Gln substitutions led to a full blue-shift of the Soret peak, whereas the Asp260(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 Asp260(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 Asp260(94), Met409(243) and Glu408(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.
University of Brussels, Nivelles, Belgium.
A
A
Jacquet
L
L
Garcia-Quintana
V
V
Deleersnyder
R
R
Fenna
A
A
Bollen
N
N
Moguilevsky
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009195:Peroxidase / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006418:Heme; D009195:Peroxidase