Purification and characterization of the nocardial acetylesterase involved in 2-butanone degradation.
An inducible acetylesterase (EC 3.1.1.6) that hydrolyzes ethyl acetate, an intermediate in the degradation of 2-butanone by Nocardia strain LSU-169, was purified. The polypeptide molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 39,500, and the enzyme molecular weight determined by sucrose density gradient centrifugation was 84,000. The purified enzyme demonstrated aggregation in polyacrylamide gels. The esterase hydrolyzed p-nitrophenyl acetate, ethyl acetate, and methyl acetate; however, enzymatic hydrolysis of phosphates, sulfates, dipeptides, lactones, or the ethyl esters of N-benzoyl-l-tyrosine could not be detected. The apparent K(m) for esterase activity with p-nitrophenyl acetate as the substrate was 6.7 x 10(-5) M, and the maximal velocity (V) was 1,223 mumol/min per mg of protein at 30 C. With ethyl acetate as the substrate, the apparent K(m) was 3.6 x 10(-4) M and V was 1,026 mumol/min per mg of protein. No significant inhibition of esterase activity was obtained with organophosphates, mercuric compounds, eserine sulfate, sodium arsanilate, NaF, CaCl(2), CoCl(2), or MnCl(2). At concentrations from 7 x 10(-4) to 4 x 10(-3) M, 2-butanol and primary alcohols with chain lengths of four or more carbons inhibited esterase activity from 59 to 86%. Linear noncompetitive inhibition of esterase activity by 3-methyl-1-butanol with a K(i) of 1.0 x 10(-3) M was demonstrated.
D000115:Acetylesterase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D000438:Alcohols / Q000494:pharmacology; D000645:Ammonium Sulfate; D002074:Butanones / Q000378:metabolism*; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D004589:Electrophoresis, Disc; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006868:Hydrolysis; D008970:Molecular Weight; D009615:Nocardia / Q000201:enzymology* / Q000378:metabolism; D010455:Peptides / Q000032:analysis
D000438:Alcohols; D002074:Butanones; D010455:Peptides; D000115:Acetylesterase; D000645:Ammonium Sulfate