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

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.

Mutational Analysis of the Integral Membrane Methyltransferase Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Reveals Potential Substrate Binding Sites

“…altered substrate binding (37). Similarly, in the human sulfotransferase SULT1A1, substrate inhibition by dopamine was observed when Phe-247 was mutated to leucine and was interpreted to mean that this substitution created space for the binding of a second dopamine molecule (35). Finally, the UDPglucuronosyltransferase human UGT1A10 has an amino acid motif ( 90 FMVF 93 ) that is postulated to be involved in binding of its phenolic substrates through ring stacking with the phenylalanine residues (38).…”

Sulfation of ractopamine and salbutamol by the human cytosolic sulfotransferases

“…The sulfation of pNP by SULT1A1 displayed a slightly positive cooperativity at low concentrations and substrate inhibition profiles at high concentrations (52). For SULT1A3, a molecular modelling analysis suggested the potential superposition of another dopamine molecule in the substrate-binding pocket and kinetic assay showed the substrate inhibition profile at high concentrations of dopamine (53). In our initial kinetic experiments, sigmoidal curve analyses were performed using Hill function at concentration ranges of 0.5 50 mM for ractopamine and 5 1,000 mM for salbutamol to investigate any possible cooperativity of the sulfation of ractopamine or salbutamol by SULT1A3.…”

Characterization of phase-II conjugation reaction of polycyclic aromatic hydrocarbons in fish species: Unique pyrene metabolism and species specificity observed in fish species

“…HPyS activity demonstrated non-hyperbolic kinetics (Figure 9), fitting with a substrate inhibition model. Some types of SULTs showed substrate inhibition for certain substrates such as human SULT1A3 and dopamine (Wang et al, 2007;Barnett et al, 2004). However, when inhibited, the velocity decreased to a plateau rather than to zero.…”