Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase.
13C and 2H kinetic isotope effects have been used to investigate the mechanism of enzymic biotin carboxylation. D(V/K) is 0.50 in 80% D2O at pD 8.0 for the forward reaction and 0.57 at pD 8.5 for the phosphorylation of ADP by carbamoyl phosphate. These values approach the theoretical maximum limit for a reaction in which a proton is transferred from a sulfhydryl to a nitrogen or oxygen base. Therefore, it appears that this portion of the reaction is at or near equilibrium. 13(V/K) at pH 8 is 1.007; the small magnitude of this number suggests that the reaction is almost fully committed by the time the carbon-sensitive steps are reached. There does not appear to be a reverse commitment to the reaction under the conditions in which 13(V/K) was determined. A large forward commitment is consistent with the failure to observe positional isotope exchange from the beta gamma-bridge position to the beta-nonbridge position in [18O4]ATP or washout of 18O from the gamma-nonbridge positions. Transfer of 18O from bicarbonate to inorganic phosphate in the forward reaction was clearly observed, however. These observations suggest that biotin carboxylase exists in two distinct forms which differ in the protonation states of the two active-site bases, one of which is a sulfhydryl. Only when the sulfhydryl is ionized and the second base protonated can catalysis take place. Carboxylation of biotin is postulated to occur via a pathway in which carboxyphosphate in formed by nucleophilic attack of bicarbonate on ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism; D002247:Carbon Isotopes; D019731:Carbon-Nitrogen Ligases; D002384:Catalysis; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012997:Solvents
D001639:Bicarbonates; D002247:Carbon Isotopes; D012997:Solvents; D000255:Adenosine Triphosphate; D003903:Deuterium; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase