Purification and properties of the P2 primary alkylsulphohydrolase of the detergent-degrading bacterium pseudomonas C12B

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Abstract
The P2 primary alkylsulfohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (200 to 250-fold) by column chromatography on DEAE-cellulose, Sephadex G-100 and butyl-agarose. The intact protein is a dimer with a MW of 160,000. Activity towards primary alkyl sulfate esters was maximal at pH 8.3, varied little in the range pH 7.8-8.7, but decreased sharply at higher pH. For a homologous series of primary alkyl sulfate substrates (C6-C12), log Km decreased linearly with increasing chain length, corresponding to a contribution to the free energy of association between enzyme and substrate of -2.5 kJ/mol for each additional CH2 group in the alkyl chain, log Ki for the competitive inhibition by secondary alkyl 2-sulfate esters followed a similar pattern (-2.4 kJ/mol for each additional CH2 group) except that only n-1 carbon atoms effectively participate in hydrophobic bonding, implying that the C-1 methyl group is not involved. Log Ki values for inhibition by primary alkanesulfonates also depended linearly on chain length but with a diminished gradient, indicating a free-energy increment of -1.2kJ/mol per additional CH2 group. The collective results showed the presence of a hydrophobic site on the enzyme capable of accommodating an alkyl chain of considerable length. Cationic structures (in the form of arginine, lysine or histidine), whose presence might be expected for binding the anionic sulfate group, were not detectable at the active site.