Carbamate kinase from Enterococcus faecalis and Enterococcus faecium

Carbamate kinase (CK) catalyzes the reversible reaction NH 2 COO − + ATP NH 2 COOPO.es2−.rb.ei3.rb + ADP, serving to synthesize ATP from carbamoyl phosphate in those microorganisms that derive energy from anaerobic arginine degradation via the arginine dihydrolase pathway. We report here the cloning and sequencing of the CK gene from Enterococcus faecalis and Enterococcus faecium and we demonstrate that the amino acid sequence of CK is identical in the two species. The enzyme, expressed and isolated from Escherichia coli using simple purification procedures, was used to generate crystals suitable for X‐ray studies and to investigate the utilization by CK of bicarbonate and other carbamate analogs. CK had a bicarbonate‐dependent ATPase activity and, therefore, is able to synthesize carboxyphosphate, an unstable compound that is an intermediate in the reactions catalyzed by carbamoyl‐phosphate synthetase (CPS) and by biotin carboxylase. Other functional similarities with CPS include the utilization of acetate by CK with a similarly high K m and the similar K m values of CK for carbamate and of CPS for bicarbonate. Enterococcal CK was inhibited by adenosine(5′)pentaphospho(5′)adenosine (Ap 5 A) and Ap 6 A and, less powerfully, by Ap 4 A, whereas Ap 3 A is essentially non‐inhibitory. Thus, inhibition by Ap 5 A seems not to be a valid criterion to differentiate between CK and CPS, for the two enzymes can be inhibited by Ap 5 A. All these results support the relatedness of CK and CPS. Finally, we used limited proteolysis : (a) to localize the epitopes for monoclonal antibodies obtained against CK; (b) to demonstrate the importance of the C‐terminus for enzyme activity; and (c) to show that Arg158 is highly exposed and may be essential for activity. Comparison of the sequence of CK with known protein sequences demonstrates considerable similarity of CK with bacterial N ‐acetylglutamate kinases, strongly suggesting that these two enzymes may share a similar structure and the same catalytic mechanism.