bi201276r_si_001.pdf (57.47 kB)
Activation and Inhibition of Pyruvate Carboxylase from Rhizobium etli
journal contribution
posted on 2011-11-15, 00:00 authored by Tonya
N. Zeczycki, Ann L. Menefee, Sarawut Jitrapakdee, John C. Wallace, Paul V. Attwood, Martin St. Maurice, W. Wallace ClelandWhile crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable
positioning of the essential activator, Mg2+, and nonessential
activator, acetyl-CoA, an understanding of how they affect catalysis
remains unclear. The current steady-state kinetic investigation indicates
that both acetyl-CoA and Mg2+ assist in coupling the MgATP-dependent
carboxylation of biotin in the biotin carboxylase (BC) domain with
pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial
velocity plots of free Mg2+ vs pyruvate were nonlinear
at low concentrations of Mg2+ and a nearly complete loss
of coupling between the BC and CT domain reactions was observed in
the absence of acetyl-CoA. Increasing concentrations of free Mg2+ also resulted in a decrease in the Ka for acetyl-CoA. Acetyl phosphate was determined to be a suitable
phosphoryl donor for the catalytic phosphorylation of MgADP, while
phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl
phosphate (Ki = 0.026 mM) and pyruvate
carboxylation (Ki = 2.5 mM). In conjunction
with crystal structures of T882A R. etli PC mutant
cocrystallized with phosphonoacetate and MgADP, computational docking
studies suggest that phosphonoacetate could coordinate to one of two
Mg2+ metal centers in the BC domain active site. Based
on the pH profiles, inhibition studies, and initial velocity patterns,
possible mechanisms for the activation, regulation, and coordination
of catalysis between the two spatially distinct active sites in pyruvate
carboxylase from R. etli by acetyl-CoA and Mg2+ are described.