Carbamate kinase: New structural machinery for making carbamoyl phosphate,the common precursor of pyrimidines and arginine

The enzymes carbamoyl phosphate synthetase (CPS) and carbamate kinase (CK) make carbamoyl phosphate in the same way: by ATP-phosphorylation of carbama te. The carbamate used by CK is made chemically, whereas CPS itself synthes izes its own carbamate in a process involving the phosphorylation of bicarb onate. Bicarbonate and carbamate are analogs and the phosphorylations are c arried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit t hat was believed to resemble the 40 kDa regions of CPS. Such belief is disp roven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and pre sents a new type of fold possibly shared by homologous acylphosphate-making enzymes. A molecular 16-stranded open beta-sheet surrounded by alpha-helic es is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active ce nter. Intersubunit interactions are very large and involve a central hydrop hobic patch and more hydrophilic peripheral contacts. The crevice holds a s ulfate that may occupy the site of an ATP phosphate, and is lined by conser ved residues. Site-directed mutations tested at two of these residues inact ivate the enzyme. These findings support active site location in the crevic e. The orientation of the crevices in the dimer precludes their physical co operation in the catalytic process. Such cooperation is not needed in the C K reaction but is a requirement of the mechanism of CPSs.