Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase: mutagenesis at metal site 2.

Phosphoenolpyruvate (PEP) carboxykinases harbor two divalent metal-binding sites. One cation interacts with the enzyme (metal binding site 1) to elicit activation, while a second cation (metal binding site 2) interacts with the nucleotide to serve as the metal nucleotide substrate. Mutants of Anaerobiospirillum succiniciproducens PEP carboxykinase have been constructed where Thr249 and Asp262, two residues of metal binding site 2 of the enzyme, were altered. Binding of the 3'(2')-O-(N-methylantraniloyl) derivative of ADP provides a test of the structural integrity of these mutants. The conservative mutation (Asp262Glu) retains a significant proportion of the wild type enzymatic activity. Meanwhile, removal of the OH group of Thr249 in the Thr249Ala mutant causes a decrease in V(max) by a factor of 1.1 x 10(4). Molecular modeling of wild type and mutant enzymes suggests that the lower catalytic efficiency of the Thr249Ala enzyme could be explained by a movement of the lateral chain of Lys248, a critical catalytic residue, away from the reaction center.

Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase.

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn2+ as the activating metal ion. The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol. 4, 990-994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3-6 orders of magnitude lower values of Vmax/Km, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased Km values for Mn2+ and PEP as compared with wild-type enzyme, suggesting a role of His225 in Mn2+ and PEP binding. From 1.5-1.6 Kcal/mol lower affinity for the 3'(2')-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His225 and Asp263 in nucleotide binding.

The effect of active site mutations in the oxaloacetate decarboxylase and pyruvate kinase-like activities of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase.

Anaerobiospirillum succiniciproducens His225Gln, Asp262Asn, Asp263Asn, and Thr249Asn phosphoenolpyruvate carboxykinases were analyzed for their oxaloacetate decarboxylase, and pyruvate kinase-like activities. The His225Gln and Asp263Asn enzymes showed increased Km values for Mn2+ and PEP compared with the native enzyme, suggesting a role of His225 and Asp263 in Mn2+ and PEP binding. No mayor alterations in Km values for oxaloacetate were detected for the varied enzymes. Alterations of His225, Asp262, Asp263, or Thr249, however, did not affect the Vmax of the secondary activities as much as they affected the Vmax for the main reaction. The results presented in this communication suggest different rate-limiting steps for the primary reaction and the secondary activities.