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J Biol Chem, Vol. 274, Issue 18, 12284-12288, April 30, 1999

Amino Acid Residues Forming the Active Site of Arylsulfatase A
ROLE IN CATALYTIC ACTIVITY AND SUBSTRATE BINDING

From the Zentrum für Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universität Göttingen, Gosslerstrasse 12d, 37073 Göttingen, Germany

Arylsulfatase A belongs to the sulfatase family whose members carry a C-formylglycine that is post-translationally 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 divalent cation and a number of polar residues, tightly interconnected 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 conservatively by either asparagine or aspartate. In all mutants V_max was decreased to 1-26% of wild type activity. The K_m was more than 10-fold increased in K123A and K302A and up to 5-fold in the other mutants. In all mutants the pH optimum was increased from 4.5 by 0.2-0.8 units. These results indicate that each of the nine residues 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.

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