Purification and characterization of the short-chain alkylsulphatase of coryneform B1a.
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.
Matts
P J
PJ
School of Molecular and Medical Biosciences, Biochemistry Unit, University of Wales College of Cardiff, U.K.
White
G F
GF
Payne
W J
WJ
D000192:Actinomycetales / Q000145:classification / Q000201:enzymology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D002384:Catalysis; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D013429:Sulfatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*
D001426:Bacterial Proteins; D013429:Sulfatases