Purification and characterization of the short-chain alkylsulphatase of coryneform B1a

Author:

Matts P J1,White G F1,Payne W J2

Affiliation:

1. School of Molecular and Medical Biosciences, Biochemistry Unit, University of Wales College of Cardiff, P0 Box 911, Cardiff CF1 3US, U.K.

2. Department of Microbiology, University of Georgia, Athens, GA 30602-2605, U.S.A.

Abstract

Using a combination of streptomycin sulphate precipitation, and DEAE-cellulose and butyl-agarose chromatography, an alkylsulphatase active towards short-chain alkyl sulphates has been purified approx. 70-fold from extracts of coryneform B1a grown on butyl-1-sulphate. The enzyme protein is dimeric with a subunit molecular mass of 77.6 kDa, has an isoelectric point of pI 7.2, and converts butyl-1-sulphate stoichiometrically into butan-1-ol and inorganic sulphate. Stoichiometric incorporation of 18O from H2(18)O into sulphate during the reaction showed that enzymic hydrolysis occurred at the O-S bond of the C-O-S ester linkage. The enzyme was active on C3-C7 linear primary alkyl sulphates but not on higher (C8,9) or lower (C1,2) homologues, although the latter pair were competitive inhibitors. The specificity constant (kcat./Km) was highest for pentyl sulphate (Km 1.89 +/- 0.38 mM; kcat. 6.86 +/- 0.52 s-1) and decreased for higher and lower homologues. No activity was detected towards C3-C9 racemic alkyl-2-sulphates, D- or L-enantiomers of butyl-2-sulphate, the symmetrical secondary alkyl sulphates pentyl-3-sulphate, heptyl-4-sulphate, nonyl-5-sulphate, C1-C8 alkane sulphonates, choline sulphate, or butyric acid-4-sulphate; none of these compounds (except the symmetrical esters and butyric acid-4-sulphate, which were not tested) was demonstrably inhibitory. The enzyme was compared with other alkylsulphatases in terms of substrate specificity and mode of action.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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