The purification and characterization of a novel D(−)-specific carbamoylase enzyme from an Agrobacterium sp. |
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Authors: | Ariel Louwrier Christopher J Knowles |
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Institution: | Biological Laboratory, University of Kent at Canterbury, Kent, United Kingdom |
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Abstract: | A carbamoylase enzyme was purified from a cell-free extract of Agrobacterium sp. with an overall yield of 81%. It was judged to be homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight of 38,000 daltons. Further studies on the native enzyme suggested that the active enzyme was present as a dimer, with a pI of 5.5. It was able to cleave a variety of N-carbamoyl substrates, but was strictly D(?) specific. It was found to have a Km of 0.82 m
and a Vmax of 31 U mg?1 for D(?) N-carbamoyl hydroxyphenylglycine in the presence of 10 m
dithiothreitol. It showed no metal ion requirements but was inhibited by iodoacetic acid and iodoacetamide, both thiol reagents. The N-terminal amino acid sequence of the enzyme was elucidated. |
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Keywords: | Carbamoylase hydantoinase N-carbamoyl hydroxyphenylglycine hydroxyphenylglycine hydroxyphenyl hydantoin |
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