Catalytic mechanism of biotin carboxylase: steady-state kinetic investigations.
Biotin carboxylase was purified from Escherichia coli by a new procedure, and its steady-state kinetic parameters were examined. MgATP and bicarbonate add to the enzyme randomly, followed by addition of biotin. Both bicarbonate and MgATP add in rapid equilibrium. A catalytic base with a pK of 6.6 is observed in V/K profiles. Inactivation studies also revealed a sulfhydryl group in the active site that is essential for catalysis. It is proposed that the acid-base catalysts are necessary for the tautomerization of biotin, which presumably enhances its nucleophilicity toward the carboxyl group donor. A second enzymic group with a pK of 6.6, whose role is unknown, is seen in Vmax profiles. The pH profiles for the biotin carboxylase catalyzed phosphorylation of ADP by carbamoyl phosphate have the same shape as the profiles for the forward reaction, which demonstrates that the enzymic bases assume the same protonation states for catalysis of transphosphorylation in either direction. The lack of reactivity of thionucleotide analogues of ATP when Mg is used as the divalent metal ion suggests that both metal ions required for reaction coordinate to the nucleotide. The second metal ion appears to be absolutely required for reaction and not merely an activator of the reaction. Characterization of a bicabonate-dependent biotin-independent ATPase activity strongly suggests that carboxylation proceeds via a carboxyphosphate intermediate.
Tipton
P A
PA
Department of Biochemistry, University of Wisconsin, Madison 53706.
Cleland
W W
WW
D000251:Adenosine Triphosphatases / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism
D013873:Thionucleotides; D000251:Adenosine Triphosphatases; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase