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Thr268 in substrate binding and catalysis in P450BM-3

G Truan1, J A Peterson

  • 1Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235-9038, USA.

Archives of Biochemistry and Biophysics
|January 24, 1998
PubMed

Insights

The P450BM-3 enzyme

Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Cytochrome P450 proteins (P450) are crucial enzymes involved in monooxygenation reactions across all domains of life.
  • P450BM-3 from Bacillus megaterium is a self-sufficient enzyme with both P450 and reductase domains, known for fatty acid hydroxylation and epoxidation.
  • Understanding P450 enzyme mechanisms is vital for drug metabolism and biosynthesis pathways.

Purpose of the Study:

  • To investigate the role of the I-helix residue T268 in the catalytic mechanism of P450BM-3.
  • To analyze the impact of the T268A active-site mutation on fatty acid oxidation and substrate binding.
  • To elucidate how specific residues influence substrate positioning and reaction coupling.

Main Methods:

  • Site-directed mutagenesis was used to create the T268A active-site mutant of P450BM-3.
  • Enzyme kinetics were performed to analyze arachidonic acid and palmitic acid oxidation.
  • NADPH consumption and product formation were measured to determine reaction coupling.
  • Cumene hydroperoxide-driven reactions were assessed to differentiate mechanisms.
  • Fatty acid binding assays were conducted to evaluate substrate interaction.

Main Results:

  • The T268A mutation significantly altered the NADPH consumption to product formation coupling ratio for both arachidonic acid and palmitic acid.
  • Cumene hydroperoxide-dependent reactions catalyzed by P450BM-3 were not affected by the T268A mutation.
  • Fatty acid binding studies indicated altered substrate interactions in the T268A mutant.
  • These findings suggest T268 plays a critical role in orienting fatty acid substrates for efficient hydroxylation.

Conclusions:

  • The I-helix residue T268 is essential for the proper positioning of fatty acid substrates during P450BM-3 catalysis.
  • Mutation of T268 affects the enzyme's coupling efficiency, highlighting its role in the catalytic cycle.
  • The results support a model where T268 actively participates in maintaining substrate orientation for productive hydroxylation.

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