Enhanced oxidation of aniline derivatives by two mutants of cytochrome c peroxidase at tryptophan 51

• Published in: The Journal of biological chemistry Vol. 268; no. 27; pp. 20037 - 20045
• Main Authors: ROE, J. A, GOODIN, D. B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25-09-1993
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Aniline Compounds - metabolism; Biological and medical sciences; Cloning, Molecular; Cytochrome c Group - metabolism; Cytochrome-c Peroxidase - biosynthesis; Cytochrome-c Peroxidase - isolation & purification; Cytochrome-c Peroxidase - metabolism; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Kinetics; Mitochondria - enzymology; Models, Molecular; Mutagenesis, Site-Directed; Oxidoreductases; Protein Conformation; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Software; Substrate Specificity; Thermodynamics; Tryptophan; Enzyme; Escherichia coli; Peroxidases; Enzymatic activity; Aniline; Structure activity relation; Bacteria; Thermodynamic analysis; Oxidation; Oxidoreductases; Mutation; Cytochrome-c peroxidase; Enterobacteriaceae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(20)80691-7

Two hyperactive mutants of cytochrome c peroxidase (CCP), W51F and W51A, catalyze the enhanced oxidation of a number of substituted
anilines. The reaction of CCP compound ES with mesidine is biphasic, while similar reactions using compound II give monophasic
kinetics. These data, in addition to the ratio of the Fe4+ = O and free-radical species observed during steady-state turnover,
indicate that reduction of the Trp-191 free radical of compound ES is more rapid than the reduction of the Fe4+ = O species.
Transient kinetics were examined for the oxidation of eight mono-substituted anilines by CCP, W51F, and W51A. Each of the
aniline derivatives were oxidized by the mutants at rates that exceeded that of the wild-type enzyme, and the rate constant
for m-chloroaniline was 400-fold faster for W51F than for wild-type CCP. Variations in the rate constants for the different
substrates follow a linear free-energy relationship using the Hammet substituent effect parameter sigma +, implicating electron
transfer from the aniline ring in the transition state. For aniline oxidation, the free energy of activation is 3 kcal/mol
lower for the mutants than for wild-type CCP, and this is due primarily to an increase in the activation entropy. These results
indicate that the enhanced kinetics of W51F and W51A result from a generalized increase in enzyme reactivity characterized
by an exo-entropic transition state such as dissociation of bound H2O from the Fe4+ = O center.