Topological mapping of the cysteine residues of N-carbamyl-D-amino-acid amidohydrolase and their role in enzymatic activity.
The N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter NRRL B11291, the enzyme used for the industrial production Of D-amino acids, was cloned, sequenced, and expressed in Escherichia coli. The protein, a dimer constituted by two identical subunits of 34,000 Da with five cysteines each, was susceptible to aggregation under oxidizing conditions and highly sensitive to hydrogen peroxide. To investigate the role of the cysteines in enzyme stability and activity, mutant proteins were constructed by site-directed mutagenesis in which the five residues were substituted by either Ala or Ser. Only the mutant carrying the Cys172 substitution was catalytically inactive, and the other mutants maintained the same specific activity as the wild type enzyme. The crucial role of Cys172 in enzymatic activity was also confirmed by chemical derivatization of the protein with iodoacetate. Furthermore, chemical derivatizations using both acrylamide and Ellman's reagent revealed that (i) none of the five cysteines is engaged in disulfide bridges, (ii) Cys172 is easily accessible to the solvent, (iii) Cys193 and Cys250 appear to be buried in the protein core, and (iv) Cys243 and Cys279 seem to be located within or in proximity of external loops and are derivatized under mild denaturing conditions. These data are discussed in light of the possible mechanisms of enzyme inactivation and catalytic reaction.
Grifantini
R
R
Eniricerche S.p.A., Genetic Engineering and Microbiology Laboratories, San Donato Milanese, Milan, Italy.
Pratesi
C
C
Galli
G
G
Grandi
G
G
D000409:Alanine; D000581:Amidohydrolases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D017931:DNA Primers; D005798:Genes, Bacterial; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids; D017354:Point Mutation; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012231:Rhizobium / Q000201:enzymology*; D012694:Serine
D017931:DNA Primers; D007461:Iodoacetates; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012694:Serine; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase; D003545:Cysteine; D000409:Alanine; D019807:Iodoacetic Acid