The crystal structure of ATP-dependent phosphoenolpyruvate carboxykinase (ATP-oxaloacetate carboxy-lyase, (transphosphorylating), E.C. 4.1.1.49; PCK) from Escherichia coli K12 has been determined using a combination of multiple isomorphous replacement and density modification, and refined to a R-index of 0.202 (R-free = 0.244) at 1.9 A resolution. PCK catalyses the decarboxylation and ATP-dependent phosphorylation of oxaloacetate to form phosphoenolpyruvate, the first committed step of gluconeogenesis in E. coli. Each PCK molecule consists of a 275 residue N-terminal and 265 residue C-terminal ar mononucleotide-binding domain, with the active site located within a cleft between the two domains. PCK is an open-faced, mixed $\alpha/\beta$ protein with a unique overall tertiary structure. The putative phosphate-binding region of the ATP-binding site adopts the P-loop motif common to many ATP- and GTP-binding proteins. However, the รข-sheet topology of the mononucleotide-binding fold of PCK differs from all other families within the P-loop containing nucleoside triphosphate hydrolase superfamily, suggesting PCK represents the first member of a new family of such proteins. The mononucleotide-binding domain also differs in structure from the classical mononucelotide-binding fold (CMBF), common to adenylate kinase, RecA, p21$\sp{{Ha}-ras}$, and elongation factor-Tu. Several highly-conserved amino acid residues among the ATP-dependent PCK family, including R65, Y207, K212, K213, H232, K254, T255, D269, K288 and R333 appear to make up the active site of the enzyme. A cysteine residue, C233, is located near the active site, and in the E. coli enzyme this residue is buried and is probably not involved in substrate binding or catalysis. Previous chemical modification studies, on several ATP- and GTP-dependent PCKs, have been assessed in view of these structural results. A mechanism of catalysis based on these and additional results is proposed. The structure of E. coli PCK complexed with the calcium-analogue Tb$\sp{3+}$ has been refined to an R-index of 0.205 (R-free = 0.259) at 2.5 A. Two binding sites for Tb$\sp{3+}$ have been determined, one within the active site coordinating to the side chains of K213, H232, and D269, and the other within the C-terminal domain, coordinating to the side chains of E508 and E511. No large structural movements are observed in PCK as a result of Tb$\sp{3+}$ binding, even though Ca$\sp{2+}$ is a known activator.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10212004-000610 |
| Date | 01 January 1996 |
| Creators | Matte, Alan Michael |
| Contributors | Delbaere, Louis T. J. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-10212004-000610 |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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