Abstract
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.
Highlights
The Fe4+=0and free-radical species observed during steady-state turnover, indicate that reduction of the cytochrome c peroxi- peroxidase (CCP) + Hz02 k p E S + Hz0
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
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 boundH20from the Fe4+=0center
Summary
We have examined ing the oxidation state of the heme, is constant and equal to the reaction of mesidine with compound I1of CCP(MKT), 100 f 10% of that expected for the enzyme in the oxyferryl which contains the oxyferryl (Fe4+=O)center without the state. Under these steady-state turnover conditions, samples Trp-191 free radical. Prepared by reductive titration of CCP-Fe3+in an anaerobic These data indicate that thereaction is limited by the reduc- electrochemical cell using methyl viologen asan electron ing substrate, and that k!PP and k;PP are of similar magnitude, transfer mediator (Goodin and McRee, 1993). Compound I1 was generated by addition of a slight transient kinetics for the reaction of mesidine with the oxi- excess (1.1equivalents) of H202to a 10 PM solution of CCP-
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