Active Site Mutations and Substrate Inhibition in Human Sulfotransferase 1A1 and 1A3

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 seque...

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Bibliographic Details
Published in:The Journal of biological chemistry 2004-04, Vol.279 (18), p.18799-18805
Main Authors: Barnett, Amanda C, Tsvetanov, Sergey, Gamage, Niranjali, Martin, Jennifer L, Duggleby, Ronald G, McManus, Michael E
Format: Article
Language:English
Subjects:
Arylsulfotransferase
Binding Sites - genetics
Dopamine - metabolism
Enzyme Inhibitors
Humans
Kinetics
Mutagenesis, Site-Directed
Mutation, Missense
Nitrophenols - metabolism
Protein Binding
Sulfotransferases - antagonists & inhibitors
Sulfotransferases - genetics
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Summary: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 ( p NP) 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 p NP as the substrate but not with dopamine. For SULT1A3, substrate inhibition is found for dopamine but not with p NP. 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M312253200