Abstract
Site-specific mutagenesis was used to replace the N-terminal cysteine in human asparagine synthetase by an alanine. The mutant enzyme was expressed in the yeast Saccharomyces cerevisiae, and the asparagine synthetase activity was analyzed in vitro. The mutation resulted in the loss of the glutamine-dependent asparagine synthetase activity, while the ammonia-dependent activity remained unaffected. These results confirm the existence of a glutamine amidotransfer domain with an N-terminal cysteine essential for the glutamine-dependent asparagine synthetase activity.
Publication types
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Comparative Study
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Alanine
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Amidophosphoribosyltransferase / genetics
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Amino Acid Sequence
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Aspartate-Ammonia Ligase / genetics*
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Aspartate-Ammonia Ligase / metabolism
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Bacillus subtilis / enzymology
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Bacillus subtilis / genetics
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Base Sequence
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Cloning, Molecular
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Cysteine*
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Glutamine / metabolism*
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Humans
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Ligases / genetics*
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Molecular Sequence Data
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Mutation*
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Oligonucleotide Probes
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Plasmids
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Saccharomyces cerevisiae / enzymology
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Saccharomyces cerevisiae / genetics
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Sequence Homology, Nucleic Acid
Substances
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Oligonucleotide Probes
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Glutamine
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Amidophosphoribosyltransferase
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Ligases
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Aspartate-Ammonia Ligase
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Cysteine
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Alanine