Influence of phenylalanines 77 and 138 on the stereospecificity of aryl sulfotransferase IV.

Aryl sulfotransferase (AST) IV (also named tyrosine-ester sulfotransferase and ST1A1) is a major phenol sulfotransferase in the rat, and it catalyzes the sulfation of many drugs, carcinogens, and other xenobiotics that contain phenol, benzylic alcohol, N-hydroxy arylamine, and oxime functional groups. Previous work discovered a stereospecificity of AST IV toward the enantiomers of 1,2,3,4-tetrahydro-1-naphthol and varying degrees of stereoselectivity with other chiral benzylic alcohols. The studies described here were directed toward understanding the roles of specific amino acid residues at the substrate binding site in determining the stereoselectivity of this sulfotransferase isoform. Docking experiments with a homology model of AST IV revealed three amino acid residues, Phe77, Phe138, and Tyr236, that may potentially be important for interactions with substituents on the chiral carbon of a benzylic alcohol serving as a sulfuryl acceptor, thereby imparting stereoselectivity. To test this hypothesis, mutants were constructed wherein each of the above residues was substituted with alanine. Kinetic studies on the sulfation of the enantiomers of 1,2,3,4-tetrahydro-1-naphthol indicated that the stereospecificity of the sulfotransferase was altered by the substitutions of alanine for either Phe77 or Phe138, but stereospecificity was maintained by alanine substitution at Tyr236. Molecular models of the mutant enzymes interacting with enantiomers of 1,2,3,4-tetrahydro-1-naphthols and with 2-naphthol indicate that Phe77 and Phe138 provide significant steric interactions at the active site that both enhance catalytic efficiency and impart stereospecificity in molecular recognition of substrates and inhibitors.