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Roles of Arg427 and Arg472 in the Binding and Allosteric Effects of Acetyl CoA in Pyruvate Carboxylase
journal contribution
posted on 2012-10-16, 00:00 authored by Abdussalam Adina-Zada, Chutima Sereeruk, Sarawut Jitrapakdee, Tonya
N. Zeczycki, Martin St. Maurice, W. Wallace Cleland, John C. Wallace, Paul V. AttwoodMutation of Arg427 and Arg472 in Rhizobium etli pyruvate carboxylase to serine or lysine greatly increased the activation
constant (Ka) of acetyl CoA, with the
increase being greater for the Arg472 mutants. These results indicate
that while both these residues are involved in the binding of acetyl
CoA to the enzyme, Arg472 is more important than Arg427. The mutations
had substantially smaller effects on the kcat for pyruvate carboxylation. Part of the effects of the mutations
was to increase the Km for MgATP and the Ka for activation by free Mg2+ determined
at saturating acetyl CoA concentrations. The inhibitory effects of
the mutations on the rates of the enzyme-catalyzed bicarbonate-dependent
ATP cleavage, carboxylation of biotin, and phosphorylation of ADP
by carbamoyl phosphate indicate that the major locus of the effects
of the mutations was in the biotin carboxylase (BC) domain active
site. Even though both Arg427 and Arg472 are distant from the BC domain
active site, it is proposed that their contacts with other residues
in the allosteric domain, either directly or through acetyl CoA, affect
the positioning and orientation of the biotin-carboxyl carrier protein
(BCCP) domain and thus the binding of biotin at the BC domain active
site. On the basis of the kinetic analysis proposed here, it is proposed
that mutations of Arg427 and Arg472 perturb these contacts and consequently
the binding of biotin at the BC domain active site. Inhibition of
pyruvate carboxylation by the allosteric inhibitor l-aspartate
was largely unaffected by the mutation of either Arg427 or Arg472.