Arginine 177 is involved in Mn(II) binding by manganese peroxidase

Maarten D. Sollewijn Gelpke, Pierre Moenne-Loccoz, Michael H. Gold

Research output: Contribution to journalArticle

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Abstract

Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV-vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric state were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent K(m) values for Mn(II) were ~20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K(m) values for the substrates, H2O2 and ferrocyanide, and the k(cat) values for Mn(II) and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by Mn(II) were ~10-fold lower than for wild-type MnP. In addition, the K(D) values calculated from the first-order plots of MnP compound II (MnPII) reduction by Mn(II) for the mutant enzymes were ~22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by Mn(II) were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from Mn(II) to the oxidized heme apparently is not affected.

Original languageEnglish (US)
Pages (from-to)11482-11489
Number of pages8
JournalBiochemistry
Volume38
Issue number35
DOIs
StatePublished - Aug 31 1999

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manganese peroxidase
Arginine
Phanerochaete
Enzymes
Rate constants
Heme
Mutation
Glyceraldehyde-3-Phosphate Dehydrogenases
Gene encoding
Bromides
Isoenzymes
Genes
Raman scattering
Electrons
Oxidation
Kinetics

ASJC Scopus subject areas

  • Biochemistry

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Arginine 177 is involved in Mn(II) binding by manganese peroxidase. / Sollewijn Gelpke, Maarten D.; Moenne-Loccoz, Pierre; Gold, Michael H.

In: Biochemistry, Vol. 38, No. 35, 31.08.1999, p. 11482-11489.

Research output: Contribution to journalArticle

Sollewijn Gelpke, Maarten D. ; Moenne-Loccoz, Pierre ; Gold, Michael H. / Arginine 177 is involved in Mn(II) binding by manganese peroxidase. In: Biochemistry. 1999 ; Vol. 38, No. 35. pp. 11482-11489.
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title = "Arginine 177 is involved in Mn(II) binding by manganese peroxidase",
abstract = "Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV-vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric state were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent K(m) values for Mn(II) were ~20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K(m) values for the substrates, H2O2 and ferrocyanide, and the k(cat) values for Mn(II) and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by Mn(II) were ~10-fold lower than for wild-type MnP. In addition, the K(D) values calculated from the first-order plots of MnP compound II (MnPII) reduction by Mn(II) for the mutant enzymes were ~22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by Mn(II) were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from Mn(II) to the oxidized heme apparently is not affected.",
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AB - Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV-vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric state were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent K(m) values for Mn(II) were ~20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K(m) values for the substrates, H2O2 and ferrocyanide, and the k(cat) values for Mn(II) and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by Mn(II) were ~10-fold lower than for wild-type MnP. In addition, the K(D) values calculated from the first-order plots of MnP compound II (MnPII) reduction by Mn(II) for the mutant enzymes were ~22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by Mn(II) were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from Mn(II) to the oxidized heme apparently is not affected.

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