N‐Carbamoyl‐d‐amino acid amidohydrolase from Comamonas sp. E222c Purification and characterization

Abstract

N‐Carbamoyl‐d‐amino acid amidohydrolase was purified 119‐fold, with 36% overall recovery from a cell‐free extract of Comamonas sp. E222c. The purified enzyme was homogeneous as judged by SDS/PAGE. The relative molecular mass of the native enzyme was 120 000 and that of the subunit was 40 000. The purified enzyme hydrolyzed various N‐carbamoyl‐d‐amino acids to d‐amino acids, ammonia and carbon dioxide. N‐Carbamoyl‐d‐amino acids having hydrophobic groups served as good substrates for the enzyme. The Km and Vmax values for N‐carbamoyl‐d‐phenylalanine were 19.7 mM and 13.1 units/mg, respectively, and those for N‐carbamoyl‐d‐p‐hydroxyphenylglycine were 13.1 mM and 0.56 units/mg, respectively. The enzyme strictly recognized the configuration of the substrate and only the d‐enantiomer of the N‐carbamoyl amino acid was hydrolyzed. The enzyme activity was not significantly affected by N‐carbamoyl‐l‐amino acids and ammonia. The enzyme was sensitive to thiol reagents and did not require metal ions for its activity. The enzyme did not hydrolyze N‐carbamoyl‐β‐alanine or N‐carbamoyl‐dl‐aspartate suggesting that the enzyme is different from the N‐carbamoylamide‐hydrolyzing enzymes involved in the pyrimidine degradation pathway. The enzyme did not hydrolyze allantoin and allantoic acid, which are intermediates in purine degradation, N‐carbamoylsarcosine and citrulline, suggesting that it is a novel N‐carbamoylamide amidohydrolase. Copyright © 1993, Wiley Blackwell. All rights reserved