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Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes
Published on: October 3, 2019
Selectivity in post-translational biotin addition to five human carboxylases
Maria Ingaramo1, Dorothy Beckett
1Department of Chemistry and Biochemistry, Center for Biological Structure and Organization, University of Maryland, College Park, Maryland 20742, USA.
The Journal of Biological Chemistry
|November 30, 2011
Summary
Human holocarboxylase synthetase (HCS) selectively attaches biotin to carboxylases. Mitochondrial carboxylases are biotinylated faster, suggesting evolved sequences ensure biotinylation before import, influencing biotin distribution.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Holocarboxylase synthetase (HCS) is crucial for biotin-dependent carboxylase activity.
- Biotinylation involves a two-step reaction forming bio-5'-AMP and linking biotin to a lysine residue.
- Five distinct biotin-dependent carboxylases exist in humans.
Purpose of the Study:
- To investigate the selectivity of human HCS in biotinylating different carboxylase substrates.
- To understand the kinetic basis for HCS substrate discrimination.
- To explore the implications of HCS specificity on biotin distribution and carboxylase function.
Main Methods:
- Single turnover stopped flow measurements.
- Quench flow kinetics.
- Analysis of biotin transfer to minimal biotin acceptor BCCP fragments.
Main Results:
- HCS-mediated biotinylation of mitochondrial carboxylase fragments (PCC, PC, MCC) is rapid and substrate association-limited.
- Biotinylation of cytoplasmic acetyl-CoA carboxylase fragments (ACC1, ACC2) is slower and exhibits hyperbolic substrate dependence.
- Kinetics correlate with HCS accessibility, suggesting evolved sequences for efficient mitochondrial carboxylase biotinylation.
Conclusions:
- HCS exhibits substrate selectivity based on kinetic properties and accessibility.
- Mitochondrial carboxylase sequences likely evolved for rapid HCS association to ensure biotinylation prior to import.
- HCS specificity plays a role in regulating biotin distribution among various carboxylases.
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