Purification and characterization of the nocardial acetylesterase involved in 2-butanone degradation

J Bacteriol. 1974 Dec;120(3):1133-43. doi: 10.1128/jb.120.3.1133-1143.1974.

Abstract

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.

MeSH terms

  • Acetylesterase* / antagonists & inhibitors
  • Acetylesterase* / isolation & purification
  • Acetylesterase* / metabolism
  • Alcohols / pharmacology
  • Ammonium Sulfate
  • Butanones / metabolism*
  • Cell Fractionation
  • Centrifugation, Density Gradient
  • Chemical Precipitation
  • Chromatography, DEAE-Cellulose
  • Electrophoresis, Disc
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Induction
  • Hydrolysis
  • Molecular Weight
  • Nocardia / enzymology*
  • Nocardia / metabolism
  • Peptides / analysis

Substances

  • Alcohols
  • Butanones
  • Peptides
  • Acetylesterase
  • Ammonium Sulfate