CRYSTAL-STRUCTURE OF ESCHERICHIA-COLI PHOSPHOENOLPYRUVATE CARBOXYKINASE - A NEW STRUCTURAL FAMILY WITH THE P-LOOP NUCLEOSIDE TRIPHOSPHATE HYDROLASE FOLD

The crystal structure of ATP-dependent phosphoenolpyruvate carboxykina se (ATP-oxaloacetate carboxy-lyase, (transphosphorylating), E.C. 4.1.1 .49; PCK) from Escherichia coli strain K12 has been determined using a combination of multiple isomorphous replacement, density modification , and partial model phase combination, and refined to a conventional X -index of 0.204 (R(free) = 0.244) at 1.9 Angstrom resolution. Each PCK molecule consists of a 275 residue N-terminal domain and 265 residue C-terminal or mononucleotide-binding domain, with the active site post ulated to be within a cleft between the two domains. PCK is an open-fa ced, mixed alpha/beta protein, with each domain having an alpha/beta/a lpha folding topology as found in several other mononucleoside-binding enzymes. The putative phosphate-binding site of ATP adopts the P-loop motif common to many ATP and GTP-binding proteins, and is similar in structure to that found within adenylate kinase. However, the beta-she et topology within the mononucleotide-binding fold of PCK differs from all other families within the P-loop containing nucleoside triphospha te hydrolase superfamily, therefore suggesting it represents the first member in a new family of such proteins. The mononucleotide-binding d omain is also different in structure compared to the classical mononuc leotide-binding fold (CMBF) common to adenylate kinase, p21ras, and el ongation factor-Tu. Several amino acid residues, including R65, K212, K213, H232, K254, D269, K288 and R333 appear to make up the active sit e of the enzyme, and are found to be absolutely conserved among known members of the ATP-dependent PCK family A cysteine residue is located near the active-site, as has been suggested for other PCKs, although i n the E. coli enzyme C233 is buried and so is most likely not involved in substrate binding or catalysis. Two binding sites of the calcium-a nalog Tb3+ have been determined, one within the active site coordinati ng to the side-chain of D269, and the other within the C-terminal doma in coordinating to the side-chains of E508 and E511. (C) 1996 Academic Press Limited.