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

Jeffrey M. Boyd, Scott A. Ensign

Research output: Contribution to journalArticlepeer-review

18 Scopus citations


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 (pKa=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 H1D5- to fully exchanged H6-acetone. The initial rate of isotopic exchange was higher than kcat 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 Km 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.

Original languageEnglish (US)
Pages (from-to)8543-8553
Number of pages11
Issue number23
StatePublished - Jun 14 2005
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry


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