Project: Research project

Project Details


P-enolpyruvate carboxykinase and pyruvate carboxylase catalyze the
formation of oxaloacetate from three-carbon substrates (P-enolpyruvate or
pyruvate), nucleotide triphosphates (GTP or ATP) and carbon dioxide or
bicarbonate. Both reactions require one metal ion in the formation of the
nucleotide metal complex and one or more additional metal ions to form
catalytically competent complexes. Both enzymes are essential steps in the
pathway of gluconeogenesis from lactate or alanine in mammals. The
steady-state kinetic properties of these enzymes have been characterized,
however many of the important features of catalysis are poorly understood.
One purpose of these studies will be to characterize the interactions of
substrates and metals at the catalytic sites of the enzymes. The
interactions of substrates or substrate analogues with metal ions will be
measured in electron paramagnetic resonance experiments which can establish
direct metal substrate contacts and quantitate metal water and metal enzyme
contacts. The rates of formation and dissociation of enzyme-substrate
complexes will be established by substrate trapping experiments using
labeled substrates. Reversibility of catalytic steps will be estimated by
positional isotopic exchange experiments which can be quantitated by
nuclear magnetic resonance or mass spectrometry. Heavy-atom kinetic
isotope effects will be used to investigate the effects of allosteric
activation on pyruvate carboxylase.

These experiments will attempt to establish coordination of the
nucleotide-bound metal and the enzyme-bound metals in the catalytic sites
of both enzymes. Phosphobiotin or carboxyphosphate will be implicated as
the intermediate in the pyruvate carboxylase reaction. The rates of
catalytic steps and their reversibility will allow analysis of commitments
to catalysis. These can then be used to establish the mechanism by which
the allosteric activator, acetyl-CoA, activates pyruvate carboxylase and
the metal activator, Mn(II), activates P-enolpyruvate carboxykinase.
Heavy-atom kinetic isotope effects may be able to distinguish between
changes in the transition state structure and changes in rate-limiting
step(s) of pyruvate carboxylase in response to the allosteric activator.
Effective start/end date1/1/901/1/90


  • Catalysis