Anaerobiospirillum succiniciproducens Phosphoenolpyruvate Carboxykinase: Mutagenesis at Metal Site 2

Abstract

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.

Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis

“…coli , human, T . cruzi , and Anaerobiospirillium succiniciproducens [ 9 , 45 – 48 ]. The structural alignment of rat, human, and Mtb Pck predicted that Mtb Pck metal binding site 1 would be formed by residues Asp295, Lys249, and His249 and metal binding site 2 by residues Thr276 and Asp295.…”

Nature of Protein–CO2 Interactions as Elucidated via Molecular Dynamics

“…In addition to a crystal structure, there is a wealth of additional experimental data against which the present MD simulations can be compared, both to ensure the validity of the computational protocol and to clarify, verify, or extend experimental results. Experimentally studied PEPCK enzymes fall into both the ATP- and GTP-dependent categories and have been studied for species such as yeast, ,− rat, − and various bacteria. − ,,,− These diverse experimental studies have yielded valuable insight into the interactions of PEPCK with metal ions, substrates (both PEP and OAA), inhibitors, and nucleotides and have also elucidated the chemistry of phosphoryl transfer. However, the interaction of PEPCK with CO 2 has not been examined in nearly as much detail in these experiments.…”