Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/28080
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Type: Journal article
Title: Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains
Author: Polyak, S.
Chapman-Smith, A.
Mulhern, T.
Cronan Jr., J.
Wallace, J.
Citation: Journal of Biological Chemistry, 2001; 276(5):3037-3045
Publisher: Amer Soc Biochemistry Molecular Biology Inc
Issue Date: 2001
ISSN: 0021-9258
1083-351X
Statement of
Responsibility: 
Steven W. Polyak, Anne Chapman-Smith, Terrence D. Mulhern, John E. Cronan, Jr., and John C. Wallace
Abstract: Biotinylation in vivo is an extremely selective post-translational event where the enzyme biotin protein ligase (BPL) catalyzes the covalent attachment of biotin to one specific and conserved lysine residue of biotin-dependent enzymes. The biotin-accepting lysine, present in a conserved Met-Lys-Met motif, resides in a structured domain that functions as the BPL substrate. We have employed phage display coupled with a genetic selection to identify determinants of the biotin domain (yPC-104) of yeast pyruvate carboxylase 1 (residues 1075-1178) required for interaction with BPL. Mutants isolated using this strategy were analyzed by in vivo biotinylation assays performed at both 30 °C and 37 °C. The temperature-sensitive substrates were reasoned to have structural mutations, leading to compromised conformations at the higher temperature. This interpretation was supplemented by molecular modeling of yPC-104, since these mutants mapped to residues involved in defining the structure of the biotin domain. In contrast, substitution of the Met residue N-terminal to the target lysine with either Val or Thr produced mutations that were temperature-insensitive in the in vivo assay. Furthermore, these two mutant proteins and wild-type yPC-104 showed identical susceptibility to trypsin, consistent with these substitutions having no structural effect. Kinetic analysis of enzymatic biotinylation using purified Met Thr/Val mutant proteins with both yeast and Escherichia coli BPLs revealed that these substitutions had a strong effect upon Km values but not kcat. The Met → Thr mutant was a poor substrate for both BPLs, whereas the Met → Val substitution was a poor substrate for bacterial BPL but had only a 2-fold lower affinity for yeast BPL than the wild-type peptide. Our data suggest that substitution of Thr or Val for the Met N-terminal of the biotinyl-Lys results in mutants specifically compromised in their interaction with BPL.
Keywords: Saccharomyces cerevisiae; Biotin; Trypsin; Pyruvate Carboxylase; Carbon-Nitrogen Ligases; Peptide Fragments; Peptide Library; Bacterial Proteins; Escherichia coli Proteins; Transcription Factors; Repressor Proteins; Amino Acid Substitution; Biotinylation; DNA Mutational Analysis; Temperature; Protein Processing, Post-Translational; Amino Acid Sequence; Protein Conformation; Sequence Homology, Amino Acid; Kinetics; Models, Molecular; Molecular Sequence Data
Description: Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
RMID: 0020010659
DOI: 10.1074/jbc.M003968200
Appears in Collections:Molecular and Biomedical Science publications

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