THE PURIFICATION AND CHARACTERIZATION OF A NOVEL D(-)-SPECIFIC CARBAMOYLASE ENZYME FROM AN AGROBACTERIUM SP

A carbamoylase enzyme was purified from a cell-free extract of Agrobac terium sp. with an overall yield of 81%. It was judged to be homogenou s on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight of 38,000 daltons. Further studies on the na tive enzyme suggested that the active enzyme was present as a dimer, w ith a pl of 5.5. It was able to cleave a variety of N-carbamoyl substr ates, but was strictly D(-) specific. It was found to have a K-m of 0. 82 mM and a V-max of 31 U mg(-1) for D(-) N-carbamoyl hydroxyphenylgly cine in the presence of 10 mM dithiothreitol. It showed no metal ion r equirements but was inhibited by iodoacetic acid and iodoacetamide, bo th thiol reagents. The N-terminal amino acid sequence of the enzyme wa s elucidated. (C) 1996 by Elsevier Science Inc.