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https://hdl.handle.net/2440/28080
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dc.contributor.author | Polyak, S. | - |
dc.contributor.author | Chapman-Smith, A. | - |
dc.contributor.author | Mulhern, T. | - |
dc.contributor.author | Cronan Jr., J. | - |
dc.contributor.author | Wallace, J. | - |
dc.date.issued | 2001 | - |
dc.identifier.citation | Journal of Biological Chemistry, 2001; 276(5):3037-3045 | - |
dc.identifier.issn | 0021-9258 | - |
dc.identifier.issn | 1083-351X | - |
dc.identifier.uri | http://hdl.handle.net/2440/28080 | - |
dc.description | Copyright © 2007 by the American Society for Biochemistry and Molecular Biology. | - |
dc.description.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. | - |
dc.description.statementofresponsibility | Steven W. Polyak, Anne Chapman-Smith, Terrence D. Mulhern, John E. Cronan, Jr., and John C. Wallace | - |
dc.language.iso | en | - |
dc.publisher | Amer Soc Biochemistry Molecular Biology Inc | - |
dc.source.uri | http://dx.doi.org/10.1074/jbc.m003968200 | - |
dc.subject | Saccharomyces cerevisiae | - |
dc.subject | Biotin | - |
dc.subject | Trypsin | - |
dc.subject | Pyruvate Carboxylase | - |
dc.subject | Carbon-Nitrogen Ligases | - |
dc.subject | Peptide Fragments | - |
dc.subject | Peptide Library | - |
dc.subject | Bacterial Proteins | - |
dc.subject | Escherichia coli Proteins | - |
dc.subject | Transcription Factors | - |
dc.subject | Repressor Proteins | - |
dc.subject | Amino Acid Substitution | - |
dc.subject | Biotinylation | - |
dc.subject | DNA Mutational Analysis | - |
dc.subject | Temperature | - |
dc.subject | Protein Processing, Post-Translational | - |
dc.subject | Amino Acid Sequence | - |
dc.subject | Protein Conformation | - |
dc.subject | Sequence Homology, Amino Acid | - |
dc.subject | Kinetics | - |
dc.subject | Models, Molecular | - |
dc.subject | Molecular Sequence Data | - |
dc.title | Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains | - |
dc.type | Journal article | - |
dc.identifier.doi | 10.1074/jbc.M003968200 | - |
pubs.publication-status | Published | - |
dc.identifier.orcid | Polyak, S. [0000-0002-8458-5194] | - |
Appears in Collections: | Aurora harvest 2 Molecular and Biomedical Science publications |
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