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 radiobac ter NRRL B11291, the enzyme used for the industrial production of D-am ino acids, was cloned, sequenced, and expressed in Escherichia coli, T he protein, a dimer constituted by two identical subunits of 34,000 Da with five cysteines each, was susceptible to aggregation under oxidiz ing conditions and highly sensitive to hydrogen peroxide, To investiga te the role of the cysteines in enzyme stability and activity, mutant proteins were constructed by site directed mutagenesis in which the fi ve residues were substituted by either Ala or Ser, Only the mutant car rying the Cys(172) substitution was catalytically inactive, and the ot her mutants maintained the same specific activity as the wild type enz yme, The crucial role of Cys(172) in enzymatic activity was also confi rmed by chemical derivatization of the protein with iodoacetate, Furth ermore, chemical derivatizations using both acrylamide and Ellman's re agent revealed that (i) none of the five cysteines is engaged in disul fide bridges, (ii) Cys(172) is easily accessible to the solvent, (iii) Cys(193) and Cys(250) appear to be buried in the protein core, and (i v) Cys(243) and Cys(279) seem to be located within or in proximity of external loops and are derivatized under mild denaturing conditions, T hese data are discussed in light of the possible mechanisms of enzyme inactivation and catalytic reaction.