Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/11304
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Type: Journal article
Title: Biotin Protein Ligase from Saccharomyces cerevisiae
Author: Polyak, S.
Chapman-Smith, A.
Brautigan, P.
Wallace, J.
Citation: Journal of Biological Chemistry, 1999; 274(46):32847-32854
Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Issue Date: 1999
ISSN: 0021-9258
1083-351X
Abstract: Catalytically active biotin protein ligase from Saccharomyces cerevisiae (EC 6.3.4.15) was overexpressed in Escherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. coli strains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function.
Keywords: Escherichia coli
Saccharomyces cerevisiae
Diphosphates
Endopeptidases
Carbon-Nitrogen Ligases
Peptide Fragments
Bacterial Proteins
Escherichia coli Proteins
Fungal Proteins
Recombinant Proteins
Transcription Factors
Repressor Proteins
Genetic Complementation Test
Sequence Deletion
Protein Binding
Kinetics
DOI: 10.1074/jbc.274.46.32847
Published version: http://dx.doi.org/10.1074/jbc.274.46.32847
Appears in Collections:Aurora harvest 2
Biochemistry publications

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