A radical on the Met-Tyr-Trp modification required for catalase activity in catalase-peroxidase is established by isotopic labeling and site-directed mutagenesis.
A transient tyrosyl-like radical with a narrow doublet X-band EPR signal is present during catalase turnover by Mycobacterium tuberculosis catalase-peroxidase (KatG). Labeling of KatG with beta-methylene-deuterated tyrosine causes a collapse of the doublet to a singlet, while for 3,5-ring-deuterated tyrosine-labeled enzyme, no changes occur in the EPR signal. Except for the replacement Tyr229Phe, all other single-tyrosine mutants of KatG exhibit the same narrow doublet EPR signal and catalase activity similar to that of the wild-type enzyme. These findings confirm that this catalytically competent radical is associated with Tyr229, whose 3' and 5' protons are replaced as a result of cross-links with neighboring Met255 and Trp107 side chains in the post-translationally modified enzyme containing a distal-side Met255-Tyr229-Trp107 adduct.
Zhao
Xiangbo
X
Department of Chemistry, Brooklyn College, 2900 Bedford Avenue, Brooklyn, New York 11210, USA.
Suarez
Javier
J
Khajo
Abdelahad
A
Yu
Shengwei
S
Metlitsky
Leonid
L
Magliozzo
Richard S
RS
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002374:Catalase / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation
D001426:Bacterial Proteins; D005609:Free Radicals; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria; D002374:Catalase