Effect of distal cavity mutations on the formation of compound I in catalase-peroxidases.
Catalase-peroxidases have a predominant catalase activity but differ from monofunctional catalases in exhibiting a substantial peroxidase activity and in having different residues in the heme cavity. We present a kinetic study of the formation of the key intermediate compound I by probing the role of the conserved distal amino acid triad Arg-Trp-His of a recombinant catalase-peroxidase in its reaction with hydrogen peroxide, peroxoacetic acid, and m-chloroperbenzoic acid. Both the wild-type enzyme and six mutants (R119A, R119N, W122F, W122A, H123Q, H123E) have been investigated by steady-state and stopped-flow spectroscopy. The turnover number of catalase activity of R119A is 14.6%, R119N 0.5%, H123E 0.03%, and H123Q 0.02% of wild-type activity. Interestingly, W122F and W122A completely lost their catalase activity but retained their peroxidase activity. Bimolecular rate constants of compound I formation of the wild-type enzyme and the mutants have been determined. The Trp-122 mutants for the first time made it possible to follow the transition of the ferric enzyme to compound I by hydrogen peroxide spectroscopically underlining the important role of Trp-122 in catalase activity. The results demonstrate that the role of the distal His-Arg pair in catalase-peroxidases is important in the heterolytic cleavage of hydrogen peroxide (i.e. compound I formation), whereas the distal tryptophan is essential for compound I reduction by hydrogen peroxide.
Regelsberger
G
G
Institute of Chemistry and the Institute of Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Jakopitsch
C
C
Rüker
F
F
Krois
D
D
Peschek
G A
GA
Obinger
C
C
D002374:Catalase / Q000378:metabolism*; D002942:Circular Dichroism; D016297:Mutagenesis, Site-Directed; D009195:Peroxidase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D002374:Catalase; D009195:Peroxidase