Amino Acid Residues Forming the Active Site of Arylsulfatase A

Abstract

Arylsulfatase A belongs to the sulfatase family whose members carry a C ¨ r Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universita ¨ttingen, Germany ¨ low, and Kurt von Figura ¨t Go ¨ttingen, -formylglycine that is post-transla- tionally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a diva- lent cation and a number of polar residues, tightly in- terconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members of the sulfatase family. To analyze their function in binding and cleaving sulfate esters, we substituted a total of nine putative active site residues of human ASA by alanine (Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150). In addition the Mg2-complexing residues (Asp29, Asp30, Asp281, and Asn282) were substituted con- servatively by either asparagine or aspartate. In all mu- tants Vmax was decreased to 1–26% of wild type activity. The Km was more than 10-fold increased in K123A and K302A and up to 5-fold in the other mutants. In all mu- tants the pH optimum was increased from 4.5 by 0.2–0.8 units. These results indicate that each of the nine resi- dues examined is critical for catalytic activity, Lys123 and Lys302 by binding the substrate and the others by direct (His125 and Asp281) or indirect participation in catalysis. The shift in the pH optimum is explained by two deprotonation steps that have been proposed for sulfate ester cleavage.