Factors that influence the translocation of the N-carboxybiotin moiety between the two sub-sites of pyruvate carboxylase.
The active site of pyruvate carboxylase, like those of all biotin-dependent carboxylases, is believed to consist of two spatially distinct sub-sites with biotin acting as a mobile carboxy-group carrier oscillating between the two sub-sites. Some of the factors that influence the location and rate of movement of the N-carboxybiotin were studied. The rate of carboxylation of the alternative substrate, 2-oxobutyrate, was measured at 0 degrees C in an assay system where the isolated enzyme--[14C]carboxybiotin was the carboxy-group donor. The results are consistent with the hypothesis that the location of the carboxybiotin in the active site is determined by the presence of Mg2+, acetyl-CoA and the oxo acid substrate. The presence of Mg2+ favours the holding of the complex at the first sub-site, whereas alpha-oxo acids induce the complex to move to the second sub-site. At low concentrations pyruvate induces this movement but does not efficiently act as a carboxy-group acceptor; hydroxypyruvate, glyoxylate and oxamate, though not carboxylated, still induce the movement. The allosteric activator acetyl-CoA exerts only a slight stimulation on the rate of translocation to the second sub-site, and this stimulation arises from an increase in the dissociation constant for Mg2+.
D000090:Acetoacetates; D000105:Acetyl Coenzyme A / Q000494:pharmacology; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D007651:Keto Acids / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D011766:Pyruvate Carboxylase / Q000378:metabolism*
D000090:Acetoacetates; D007651:Keto Acids; D046911:Macromolecular Substances; C016635:acetoacetic acid; C024582:N1'-carboxybiotin; D001710:Biotin; D000105:Acetyl Coenzyme A; D011766:Pyruvate Carboxylase; D008274:Magnesium