Escherichia coli phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxylase (transphorsphorylating) EC 4.1.1.49) catalyzes the decarboxylation and subsequent phosphorylation of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of Mg2+ATP and synergistic catalysis has been observed in the presence of Ca2+ or Mn2+. Structural analyses have shown that active site residues Arg333, Ser250 and Tyr207 are coordinated differently in E. coli PCK structures complexed with Mg2+ATP-oxalate, Mg2+ATP-Mn2+-pyruvate and Mg2+ATP-Ca2+-pyruvate; hence, we hypothesize that the function of Arg333, Ser250 and Tyr207, depends on the absence or presence of Ca2+ or Mn2+ during catalysis by E. coli phosphoenolpyruvate carboxykinase (PCK).
In order to verify this hypothesis, site directed mutagenesis of the pckA gene was used to convert Arg333 to Gln, Ser250 to Ala and Tyr207 to Phe, while 14CO2 exchange assay and x-ray crystallography were used to determine the effects of these mutations on catalysis by E. coli PCK in the presence of OAA and Mg2+ATP with Ca2+ or Mn2+ metal ions. Kinetic analysis showed that the Tyr207Phe mutation decrease kcat by 1.7 fold, while Ser250Ala and Arg333Gln reduced kcat by 10.8 and 4,555 fold respectively in the presence of Mg2+ATP and OAA. In the presence of Mg2+ATP, OAA and Ca2+, Arg333Gln, Ser250Ala and Tyr207Phe mutations reduced kcat by 11,688, 44 and 2 fold respectively. In the presence of Mg2+ATP, OAA and Mn2+ Arg333Gln, Ser250Ala and Tyr207Phe mutations reduced kcat by 2,880, 4 and 5.5 fold respectively. The crystal structure of Ser250Ala complexed with Mg2+ATP-Mn2+-pyruvate, showed that in the presence of Mn2+, Ser250Ala mutation reduced the angle between the γ-phosphate of ATP and residue 250 by 6.2 Å and increased the distance between the hydroxyl group of Tyr207 and the CH2 group of pyruvate by 0.5 Å. As a result we conclude that Arg333 is important for oxaloacetate decarboxylation and phosphorylation. During catalysis in the presence of Mg2+ATP with or without Ca2+ or Mn2+, Ser250 functions to maintain one γ-phosphate oxygen of ATP in an eclipsed conformation, while Tyr207 functions to drive oxaloacetate decarboxylation during catalysis in the presence of Mn2+ ion.
Kinetic and structural studies of E. coli PCK have previously been used to show that Asp269 is involved in metal coordination, while Lys254 and Arg65 are important for Mg2+ATP and OAA binding to E. coli PCK respectively. In this study the E. coli PCK Asp269Asn-Mg2+ATP-Ca2+-pyruvate crystal structure showed that the Asp269Asn mutation reduced the number of ligands coordinating Ca2+ from seven to three, while no electron density was observed for Mg2+ATP and OAA in Lys254Ser and Arg65Gln crystal structures respectively.
| Identifer | oai:union.ndltd.org:USASK/oai:ecommons.usask.ca:10388/ETD-2015-09-2249 |
| Date | 2015 September 1900 |
| Contributors | Hughes, Goldie |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text, thesis |
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