Purification and properties of a homogeneous aryl sulfatase A from rabbit liver.

Aryl sulfatase A (aryl sulfate sulfohydrolase EC 3.1.6.1) has been purified > 10,000-fold from rabbit liver; by disc gel electrophoresis the enzyme appears homogeneous. Various properties of the enzyme have been determined and comparisons are made with other aryl sulfatases. Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is made up of monomers of molecular weight ～ 70,000. At pH 7.4 the enzyme exists as a dimer whereas a tetrameric form predominates at pH 4.8.The enzyme exhibits the anomalous kinetics often observed with aryl sulfatase A from mammalian tissues (the enzyme is modified to an inactive form while degrading substrate and the inactive form can be reactivated by sulfate ion). The enzyme activity has been studied under a variety of reaction conditions. Two pH optima are observed and neither enzyme concentration or changes in ionic strength appear to have an effect on the relative magnitudes of the optima. Aryl sulfatase A is competitively inhibited by potassium sulfate, potassium phosphate, and sodium sulfite (K_i = 2.9 × 10^?3 M, 3.4 × 10^?5 M, and 1.1 × 10^?6 M, respectively). Kinetic constants for some substituted phenyl sulfate esters have been determined. The variation in V is not consistent with a reaction mechanism involving a rate-limiting breakdown of a common intermediate.The inactive (modified) form of the enzyme has been isolated from reaction mixtures containing aryl sulfatase A and substrate. A procedure is presented for determining the relative amount of modified and native enzyme in these preparations. In the presence of substrate, sulfate displaces the equilibrium between native and modified enzyme in favor of native enzyme. In the absence of substrate neither sulfate or phosphate have an effect on the equilibrium. A study is made of the temperature dependence of the process in which the modified enzyme is converted back to native enzyme. The relatively small entropy of activation for the conversion of the modified to the native form (ΔS3 = ?8 cal/mole deg) does not seem to be consistent with a major modification of protein conformation.