Purification and characterization of acetone carboxylase from Xanthobacter strain Py2.
Acetone metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an alpha2beta2gamma2 quaternary structure. The carboxylation of acetone was coupled to the hydrolysis of ATP and formation of 1 mol AMP and 2 mol inorganic phosphate per mol acetoacetate formed. ADP was also formed during the course of acetone consumption, but only accumulated at low, substoichiometric levels ( approximately 10% yield) relative to acetoacetate. Inorganic pyrophosphate could not be detected as an intermediate or product of acetone carboxylation. In the absence of CO2, acetone carboxylase catalyzed the acetone-dependent hydrolysis of ATP to form both ADP and AMP, with ADP accumulating to higher levels than AMP during the course of the assays. Acetone carboxylase did not have inorganic pyrophosphatase activity. Acetone carboxylase exhibited a Vmax for acetone carboxylation of 0.225 micromol acetoacetate formed min-1.mg-1 at 30 degrees C and pH 7.6 and apparent Km values of 7.80 microM (acetone), 122 microM (ATP), and 4. 17 mM (CO2 plus bicarbonate). These studies reveal molecular properties of the first bacterial acetone-metabolizing enzyme to be isolated and suggest a novel mechanism of acetone carboxylation coupled to ATP hydrolysis and AMP and inorganic phosphate formation.
Sluis
M K
MK
Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, USA.
Ensign
S A
SA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D002262:Carboxy-Lyases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics
D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D002262:Carboxy-Lyases; C100963:acetone carboxylase