Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation

• We have probed the roles of residues in the allosteric site of R. etli pyruvate carboxylase.
• Asp471 is critical to the allosteric activation of the enzyme by acetyl CoA.
• Mutation of Arg469, Asp1018 and Glu1027 increased acetyl CoA-independent activity.
• These residues form interactions with others that constrain enzyme activity.

We have examined the roles of Asp1018, Glu1027, Arg469 and Asp471 in the allosteric domain of Rhizobium etli pyruvate carboxylase. Arg469 and Asp471 interact directly with the allosteric activator acetyl coenzyme A (acetyl CoA) and the R469S and R469K mutants showed increased enzymic activity in the presence and absence of acetyl CoA, whilst the D471A mutant exhibited no acetyl CoA-activation. E1027A, E1027R and D1018A mutants had increased activity in the absence of acetyl CoA, but not in its presence. These results suggest that most of these residues impose restrictions on the structure and/or dynamics of the enzyme to affect activity.