ATP-dependent enolization of acetone by acetone carboxylase from Rhodobacter capsulatus

Acetone carboxylase catalyzes the carboxylation of acetone to acetoacetate with concomitant hydrolysis of ATP to AMP and two inorganic phosphates. The biochemical, molecular, and genetic properties of acetone carboxylase suggest it represents a fundamentally new class of carboxylase. As the initial step in catalysis, an α-proton from an inherently basic (pK_a=20) methyl group is abstracted to generate the requisite carbanion for attack on CO ₂. In the present study α-proton abstraction from acetone has been investigated by using gas chromatography/mass spectrometry to follow proton-deuteron exchange between De-acetone and water. Acetone carboxylase-catalyzed proton-deuteron exchange was dependent upon the presence of ATP, Mg²⁺, and a monovalent cation (K⁺, Rb⁺, NH4⁺), and produced mixtures of isotopomers, ranging from singly exchanged H₁D_5- to fully exchanged H₆-acetone. The initial rate of isotopic exchange was higher than k_cat for acetone carboxylation. The time course of isotopic exchange showed that multiple exchange events occur for each acetone-binding event, and there was a 1:1 stoichiometric relationship between molecules of ATP hydrolyzed and the sum of new acetone isotopomers formed. ADP rather than AMP was formed as the predominant product of ATP hydrolysis during isotopic exchange. The stimulation of H^+-D⁺ exchange and ATP hydrolysis by K⁺ followed saturation kinetics, with apparent K_m values of 13.6 and 14.2 mM for the two activities, respectively. The rate of H⁺ exchange into D₆-acetone was greater than the rate of D⁺ exchange into H₆-acetone. There was an observable solvent (H₂O vs D₂O) isotope effect (1.7) for acetone carboxylation but no discernible substrate (H₆- vs D₆-acetone) isotope effect. It is proposed that α-proton abstraction from acetone occurs in concert with transfer of the γphosphoryl group of ATP to the carbonyl oxygen, generating phosphoenol acetone as the activated nucleophile for attack on CO₂.