The manganese binding site of manganese peroxidase: Characterization of an Asp179Asn site-directed mutant protein
- Oregon Graduate Inst. of Science & Technology, Portland, OR (United States); and others
A site-directed mutant, D179N, in the gene encoding Phanerochaete chrysosporium manganese peroxidase isozyme 1 (mnp1), was created by overlap extension, using polymerase chain reaction. The mutant gene was expressed in P. chrysosporium under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidase (MnP) was purified, and its spectra and MW were very similar to those of the wild-type enzyme. Steady-state kinetic analysis of MnP D179N revealed that the K{sub m} for the substrate Mn{sup II} was {approximately}50-fold greater than the corresponding K{sub m} for the wild-type recombinant enzyme (3.7 mM versus {approximately}70 {mu}M). Likewise, the k{sub cat} value for Mn{sup II} oxidation of the mutant protein was only 1/265 of that for the wild-type enzyme. By comparison, the apparent K{sub m} for H{sub 2}O{sub 2} of MnP D179N was similar to the corresponding value of the wild-type MnP. The first-order rate constant for MnP D179N compound II reduction by Mn{sup II} was approximately 1/200 of that for the wild-type enzyme. The equilibrium dissociation constant (K{sub D}) for MnP D179N compound II reduction by Mn{sup II} was {approximately}100-fold greater than the K{sub D} for the wild-type compound II. In contrast, the second-order rate constant for p-cresol reduction of the mutant compound II was similar to that of the wild-type enzyme. These results also suggest that the mutation affects the binding of Mn{sup II} to the enzyme and, consequently, the rate of compound II reduction by Mn{sup II}. In contrast, the mutation apparently does not have a significant effect on H{sub 2}O{sub 2} cleavage during compound I formation or on p-cresol reduction of compound II. The results strongly suggest that Asp179 is one of the acidic amino acid ligands in the Mn{sup II} binding site of MnP. 53 refs., 7 figs., 3 tabs.
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG06-92ER20093
- OSTI ID:
- 283343
- Journal Information:
- Biochemistry (Eaton), Vol. 34, Issue 33; Other Information: PBD: 22 Aug 1995
- Country of Publication:
- United States
- Language:
- English
Similar Records
Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase
Manganese peroxidase from the basidiomycete Phanerochaete chrysosporium: spectral characterization of the oxidized states and the catalytic cycle