BACKGROUND: Phosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in 
the gluconeogenesis pathway, where it catalyzes the reversible conversion of 
oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO2. The substrates for 
Escherichia coli PEPCK are OAA and MgATP, with Mn2+ acting as a cofactor. 
Analysis of PEPCK structures have revealed amino acid residues involved in 
substrate/cofactor coordination during catalysis.
METHODS: Key residues involved in coordinating the different substrates and 
cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used 
for kinetic assays. The structure of some mutant enzymes were determined using 
X-ray crystallography.
RESULTS: Mutation of residues D269 and H232, which comprise part of the 
coordination sphere of Mn2+, reduced kcat by 14-fold, and significantly 
increased the Km values for Mn2+ and OAA. Mutation of K254 a key residue in the 
P-loop motif that interacts with MgATP, significantly elevated the Km value for 
MgATP and reduced kcat. R65 and R333 are key residues that interacts with OAA. 
The R65Q and R333Q mutations significantly increased the Km value for OAA and 
reduced kcat respectively.
CONCLUSIONS: Our results show that mutation of residues involved in coordinating 
OAA, MgATP and Mn2+ significantly reduce PEPCK activity. K254 plays an important 
role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation 
and phosphoryl transfer by E. coli PEPCK.
GENERAL SIGNIFICANCE: In higher organisms including humans, PEPCK helps to 
regulate blood glucose levels, hence PEPCK is a potential drug target for 
patients with non-insulin dependent diabetes mellitus.