Role of proximal methionine residues in Leishmania major peroxidase

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Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major
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Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

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Rajesh K Yadav1, Swati Pal, Subhankar Dolai

  • 1Division of Structural Biology and Bio-informatics, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4, Raja S.C. Mullick Road, Kolkata, India.

Archives of Biochemistry and Biophysics
|September 7, 2011
PubMed

Insights

Leishmania major peroxidase (LmP) methionine residues are crucial for controlling enzyme catalysis. Mutating these residues stabilizes the Compound I radical and alters substrate oxidation, impacting ferrocytochrome c oxidation.

Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Leishmania major peroxidase (LmP) shares structural similarities with cytochrome c peroxidase and ascorbate peroxidase.
  • The role of conserved proximal methionine residues in LmP catalysis remains uninvestigated.

Purpose of the Study:

  • To investigate the role of conserved proximal methionine residues (Met248 and Met249) in Leishmania major peroxidase (LmP) catalysis using point mutagenesis.
  • To determine the impact of these mutations on enzyme activity and the stability of reaction intermediates.

Main Methods:

  • Point mutagenesis was used to create methionine mutants of LmP.
  • Steady-state kinetics were performed to assess enzyme activity with ferrocytochrome c and small molecule substrates (guaiacol, iodide).
  • Diode array stopped-flow spectral studies were employed to analyze the stability of Compound I.

Main Results:

  • Methionine mutants exhibited <2% of wild-type ferrocytochrome c oxidation activity.
  • The rate constant for Compound I formation was unaffected in mutants.
  • Activity towards small molecule substrates (guaiacol, iodide) increased in mutants.
  • The porphyrin π-cation radical of Compound I was more stable in mutant LmP compared to wild type.

Conclusions:

  • Electronegative sulfur atoms in the proximal pocket are critical for controlling the location of the Compound I radical in heme peroxidases.
  • These residues play a significant role in the oxidation of ferrocytochrome c by LmP.

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