Covalent modification as the cause of the anomalous kinetics of aryl sulfatase A

Abstract

Mammalian aryl sulfatase A enzymes are known to exhibit an anomalous kinetic behavior in which the enzyme becomes inactivated as it catalyzes the hydrolysis of substrate. Part of the activity of this inactive, turnover-modified form of the enzyme can apparently be restored by the simultaneous presence of substrate and sulfate ion. The present experiments, conducted with 2-hydroxy-5-nitrophenyl [ 35S]sulfate (nitrocatechol sulfate), establish that the turnover-modified enzyme is covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of 35S per mole of enzyme monomer (each monomer of rabbit liver aryl sulfatase consists of two equivalent subunits). It is also shown that isolated, turnover-modified enzyme has lost 80% of its secondary structure when compared to the native enzyme. A commonly used sulfating agent, pyridine-sulfur trioxide complex brings about a similar loss of activity and of secondary structure.