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Role for an essential tyrosine in peptide amidation
Mithu De1, Joseph Bell1, Ninian J Blackburn1
1Neuroscience Department, University of Connecticut Health Center, Farmington, Connecticut 06030-3401.
The Journal of Biological Chemistry
|May 18, 2006
Summary
The peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalytic core functions as a ureidoglycolate lyase. A key tyrosine residue (Tyr654) is essential, acting as an iron ligand in a vital iron-zinc bimetallic site.
Area of Science:
- Biochemistry
- Enzymology
- Metalloprotein characterization
Background:
- Peptidylglycine alpha-amidating monooxygenase (PAM) is crucial for post-translational modification of peptides.
- The catalytic core of peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) is responsible for the alpha-amidation reaction.
- Understanding the metal ion cofactors and their roles in PAL activity is essential for elucidating its catalytic mechanism.
Purpose of the Study:
- To investigate the ureidoglycolate lyase activity of the PAL catalytic core (PALcc).
- To identify the bound metal ions within PALcc and determine their functional significance.
- To elucidate the role of a conserved tyrosine residue in PALcc activity and metal binding.
Main Methods:
- Purification of the PAL catalytic core (PALcc).
- Enzyme activity assays to assess ureidoglycolate lyase function.
- Site-directed mutagenesis of a conserved tyrosine residue (Tyr654).
- Metal ion analysis and spectrophotometric characterization (absorption at 560 nm).
Main Results:
- Purified PALcc exhibits ureidoglycolate lyase activity and contains bound calcium, zinc, and iron.
- Limiting cellular iron availability reduces PALcc specific activity.
- Mutation of Tyr654 to phenylalanine abolishes PAL activity, iron binding, and zinc binding.
- Spectroscopic data suggest a phenolate-Fe(III) charge transfer complex.
Conclusions:
- Tyr654 is essential for the catalytic activity of PALcc.
- Tyr654 likely serves as a ligand for Fe(III) within an iron-zinc bimetallic active site.
- The findings provide insights into the metalloenzyme's structure-function relationship and catalytic mechanism.
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