Abstract
Human SULT1A1 is primarily responsible for sulfonation of xenobiotics, including the activation of promutagens, and it has been implicated in several forms of cancer. Human SULT1A3 has been shown to be the major sulfotransferase that sulfonates dopamine. These two enzymes shares 93% amino acid sequence identity and have distinct but overlapping substrate preferences. The resolution of the crystal structures of these two enzymes has enabled us to elucidate the mechanisms controlling their substrate preferences and inhibition. The presence of two p-nitrophenol (pNP) molecules in the crystal structure of SULT1A1 was postulated to explain cooperativity at low and inhibition at high substrate concentrations, respectively. In SULT1A1, substrate inhibition occurs with pNP as the substrate but not with dopamine. For SULT1A3, substrate inhibition is found for dopamine but not with pNP. We investigated how substrate inhibition occurs in these two enzymes using molecular modeling, site-directed mutagenesis, and kinetic analysis. The results show that residue Phe-247 of SULT1A1, which interacts with both p-nitrophenol molecules in the active site, is important for substrate inhibition. Mutation of phenylalanine to leucine at this position in SULT1A1 results in substrate inhibition by dopamine. We also propose, based on modeling and kinetic studies, that substrate inhibition by dopamine in SULT1A3 is caused by binding of two dopamine molecules in the active site.
Highlights
The cytosolic sulfotransferase (SULT)1 gene superfamily encodes enzymes catalyzing sulfonation of numerous xeno- and endobiotics, such as drugs, hormones (e.g. 17-estradiol, thyroid hormones, and dehydroepiandrosterone), chemical carcinogens, bile acids, and neurotransmitters
The results show that residue Phe-247 of SULT1A1, which interacts with both p-nitrophenol molecules in the active site, is important for substrate inhibition
The crystal structures of mouse estrogen SULT1E1 [14], human SULT1A3 [15, 16], SULT domain of the human membrane bound heparan sulfate N-deacetylase/N-sulfotransferase 1 [17], human hydroxysteroid SULT2A1 [18, 19], and human estrogen SULT1E1 [20]; in addition, site-directed mutagenesis of critical amino acids in these enzymes has provided information on catalytic mechanism of these enzymes. This laboratory reported the structure of human SULT1A1, crystallized in the presence of 3Ј-phosphoadenosine 5Ј-phosphate (PAP) with two p-nitrophenol molecules bound in the active site [21]
Summary
The cytosolic sulfotransferase (SULT) gene superfamily encodes enzymes catalyzing sulfonation of numerous xeno- and endobiotics, such as drugs, hormones (e.g. 17-estradiol, thyroid hormones, and dehydroepiandrosterone), chemical carcinogens, bile acids, and neurotransmitters. The crystal structures of mouse estrogen SULT1E1 [14], human SULT1A3 [15, 16], SULT domain of the human membrane bound heparan sulfate N-deacetylase/N-sulfotransferase 1 [17], human hydroxysteroid SULT2A1 [18, 19], and human estrogen SULT1E1 [20]; in addition, site-directed mutagenesis of critical amino acids in these enzymes has provided information on catalytic mechanism of these enzymes This laboratory reported the structure of human SULT1A1, crystallized in the presence of 3Ј-phosphoadenosine 5Ј-phosphate (PAP) with two p-nitrophenol (pNP) molecules bound in the active site [21]. This structure is similar to the other SULT enzymes as it incorporates a core PAP binding site. Site-directed mutagenesis was performed on residues interacting with the substrate molecule at the active site of SULT1A1 indicated that the flexible residue Phe-247 is important for substrate inhibition by dopamine
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