A series of d-alkan-2-yl sulphate esters (C(7)-C(14)) were prepared by sulphation of the resolved parent alcohols by a method that entails complete retention of configuration. These sulphate esters were tested as substrates for the stereospecific CS2 secondary alkylsulphohydrolase of Comamonas terrigena. V(max.) reached a maximum with the C(9) compound, whereas logK(m) decreased linearly as the alkyl-chain length was increased from C(7) to C(14). A parallel series of l-alkan-2-yl sulphates was also prepared, and these esters, together with homologous series of primary alkyl sulphates and primary alkanesulphonates, were shown to be competitive inhibitors of the CS2 enzyme. For each series of compounds, logK(i) values decreased linearly with increasing alkyl-chain length. Plots of chain length against the standard free energy of binding (DeltaG(0)) of substrate and inhibitors to the CS2 enzyme showed that the standard free energy of association of a -CH(2)- group with the enzyme was 2.0-2.4kJ/mol for all classes of compound studied, indicating an important contribution from hydrophobic interactions to the overall binding. Plots for d-alkan-2-yl sulphate substrates and primary alkyl sulphate inhibitors were nearly coincident, suggesting that the overall interaction between a primary ester and the enzyme is the same as that between the isomeric secondary substrate and the enzyme. Plots for l-alkan-2-yl sulphate and alkanesulphonate inhibitors were very similar to each other, but were displaced by 1.5-3.0kJ/mol from that for substrate binding. This indicates that the binding of any one of these particular inhibitors involves one carbon atom fewer than the number involved in binding a substrate of the same chain length. These observations are discussed in terms of a three-point attachment of substrate to the enzyme involving the alkyl chain, sulphate group and the C-1 methyl group.
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