| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 88 === N-acetyl microperoxidase(Ac-MP-8) is a water soluble , ferric heme model for the peroxidase . In this thesis, we use stopped-flow technique, both time-resolved and fixed wavelength, to study the kinetics for the reaction of equimolar of Ac-MP-8 and H2O2. Under single-turnover condition, the rate constants for the formation of intermediate and restoration of native MP by the presence of substrate can easily be obtained.
The reaction of Ac-MP-8 with H2O2 at pH 10.7 results in a red shift of the Soret band from 397 nm to 408nm indicating the formation of compound II, however, at pH 7.09 the decreasing absorbance at 395nm suggests the formation of compound I. Upon addition of substrate, the intermediate is converted back to the native MP . The proposed catalytic pathway is:
k1 k2 k3
Fe(Ⅲ)(P)＋H2O2 → (P‧+)Fe(Ⅳ)=O＋AH2 → (P)Fe(Ⅳ)=O+AH2 → Fe(Ⅲ)(P)＋ A + AH2
From the time-resolved stopped-flow absorption experiment, it was observed that compound II dominates at high pH, while compound I dominates at low pH. The pH dependence of k1 is bell-shaped with two pka of 8.65 and 9.55, which are ascribed to, respectively , proton loss from Fe(III)MP bound H2O2 and H2O.
Because the reaction of compound I to compound II is very fast at pH 10.7, the rate constant determined from initial rate by monitoring the absorbance change at 395nm and 415nm gives the value of k1=7.95(±0.6)e4 M-1s-1 .The rate constant, k3, for the reaction of homovanillic acid and compound II at pH 10.7 is 7.62e5 M-1s-1 . Since the reaction product is fluorescent, we can also determine the rate of product formation by monitoring the increase in fluorescence intensity as the reaction proceeds. The obtained rate constant is 6.88e5 M-1s-1, which is in good agreement with k3 . Thus the value of k3 can serve as a measure of the substrate reactivity. The values of k3 have been determined for various substrates and are found to vary by three orders of magnitude. (1.7e7 M-1s-1 for 1-naphthol; 2.4e3 M-1s-1 for ferrocyanide). Microperoxidase with large peptide shows small KM because long peptide chain creates a hydrophobic environment which favors the nonpolar substrate.
Demetalation of Fe(III)MP-8 by anhydrous HF gives metal free MP-8 , which then reacts with Mn(OAc)2 to produce Mn(III)MP-8 . The observed kcat for Mn(III)MP is 300-fold smaller than that of Fe(III)MP . We have also examined the factors that affect the rates of MP-catalyzed reaction. We found that guanidinium chloride, NaCl, Na2CO3, and Tris accelerated the reaction, but methanol retarded it. These are due to the different stabilization of the reactants and the transition state. By carrying out the temperature-dependence experiments, we have determined the thermdynamic parameters of the reactions under those conditions. The results show the increase of ΔH and ΔSby the presence of guanidinium chloride, NaCl, Na2CO3, and Tris, and the decrease of ΔH and ΔS by the addition of methanol. It’s known that an increase in ΔH is unfavorable for the reactions, whereas an increase of ΔS will make the reaction favorable. The effects of ΔH and ΔS tend to cancel each other. The results show that the contribution of ΔS is larger than ΔH. Different substrates show different rate constants under identical reaction conditions. The observed rate constants decrease in the following order: 1-naphthol > 4-methoxyphenol > o-methoxyphenol > ABTS > p-anisidine > ferrocyanide. The rate constants are linearly correlated with their oxidation potentials.
|
|---|
