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. 1974 Dec;120(3):1133–1143. doi: 10.1128/jb.120.3.1133-1143.1974

Purification and Characterization of the Nocardial Acetylesterase Involved in 2-Butanone Degradation

Elizabeth F Eubanks a,1, F W Forney a,2, A D Larson a
PMCID: PMC245892  PMID: 4436255

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 Km for esterase activity with p-nitrophenyl acetate as the substrate was 6.7 × 10−5 M, and the maximal velocity (V) was 1,223 μmol/min per mg of protein at 30 C. With ethyl acetate as the substrate, the apparent Km was 3.6 × 10−4 M and V was 1,026 μmol/min per mg of protein. No significant inhibition of esterase activity was obtained with organophosphates, mercuric compounds, eserine sulfate, sodium arsanilate, NaF, CaCl2, CoCl2, or MnCl2. At concentrations from 7 × 10−4 to 4 × 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 Ki of 1.0 × 10−3 M was demonstrated.

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Selected References

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