Lack of substrate inhibition in a monomeric form of human cytosolic SULT2A1

Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA.
Hormone molecular biology and clinical investigation 12/2010; 3(1):357-366. DOI: 10.1515/HMBCI.2010.041
Source: PubMed

ABSTRACT Mammalian cytosolic sulfotransferases (SULTs) frequently show substrate inhibition during the sulfation of increasing concentrations of substrates. SULT2A1, a major human liver isoform responsible for the conjugation of hydroxysteroids, bile acids and aliphatic hydroxyl groups in drugs and xenobiotics, is a homodimer and displays substrate inhibition during the conjugation of dehydroepiandrosterone (DHEA). Maltose binding protein (MBP)-SULT2A1 fusion protein, produced as an intermediate step in the purification of the SULT2A1 homodimer, elutes during size exclusion chromatography as a monomer. The initial-rate parameters (K(m) and V(max)) of the monomer (MBP-SULT2A1) and native SULT2A1 dimer for DHEA sulfation are extremely similar; however, the monomer is not inhibited by DHEA. Intrinsic fluorescence studies show that two DHEA molecules bind each SULT2A1 subunit, one in the catalytic site and one in an apparent allosteric site. Lack of dimerization in the MBP-SULT2A1 fusion protein decreased the K(d) for binding of DHEA at the allosteric site. These results suggest that formation of the homodimer is associated with structural rearrangements leading to increased DHEA binding at an allosteric site that is associated with substrate inhibition.

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    • "This model of the involvement of SULT dimerization in substrate inhibition remains to be tested. However , it is worth noting that SULT isoforms have been reported to lose susceptibility to substrate inhibition upon monomerization (Cook et al. 2010a). "
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    ABSTRACT: The cytosolic sulfotransferases (SULTs) are dimeric enzymes that catalyze the transformation of hydrophobic drugs and hormones into hydrophilic sulfate esters thereby providing the body with an important pathway for regulating small molecule activity and excretion. While SULT dimerization is highly conserved, the necessity for the interaction has not been established. To perform its function, a SULT must efficiently bind the universal sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), and release the byproduct, 3′, 5′-diphosphoadenosine (PAP), following catalysis. We hypothesize this efficient binding and release of PAPS/PAP may be connected to SULT dimerization. To allow for the visualization of dynamic protein interactions critical for addressing this hypothesis and to generate kinetically testable hypotheses, molecular dynamic simulations (MDS) of hSULT1B1 were performed with PAPS and PAP bound to each dimer subunit in various combinations. The results suggest the dimer subunits may possess the capability of communicating with one another in a manner dependent on the presence of the cofactor. PAP or PAPS binding to a single side of the dimer results in decreased backbone flexibility of both the bound and unbound subunits, implying the dimer subunits may not act independently. Further, binding of PAP to one subunit of the dimer and PAPS to the other caused increased flexibility in the subunit bound to the inactive cofactor (PAP). These results suggest SULT dimerization may be important in maintaining cofactor binding/release properties of SULTs and provide hypothetical explanations for SULT half-site reactivity and substrate inhibition, which can be analyzed in vitro.
    06/2015; 3(3). DOI:10.1002/prp2.147
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    • "The physiological significance of sulfotransferase dimerization has been examined for a number of different enzymes. For SULT2A1, dimerization affects enzyme kinetics, substrate binding to the allosteric site and substrate inhibition [18]. By contrast, the homomeric form of SULT1A1 is structurally unstable and is more readily denatured by temperature and urea compared to the wild-type protein [19]. "
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    ABSTRACT: The cytosolic sulfotransferase SULT4A1 is highly conserved between mammalian species but its function remains unknown. Polymorphisms in the SULT4A1 gene have been linked to susceptibility to schizophrenia. There are 2 major SULT4A1 transcripts in humans, one that encodes full length protein (wild-type) and one that encodes a truncated protein (variant). Here, we investigated the expression of SULT4A1 in human tissues by RT-PCR and found the wild-type mRNA to be expressed mainly in the brain, gastrointestinal tract and prostate while the splice variant was more widely expressed. In human cell-lines, the wild-type transcript was found in neuronal cells, but the variant transcript was expressed in nearly all other lines examined. Western blot analysis only identified SULT4A1 protein in cells that expressed the wild-type mRNA. No variant protein was detected in cells that expressed the variant mRNA. Ectopically expressed full length SULT4A1 protein was stable while the truncated protein was not, having a half-life of approximately 3 hr. SULT4A1 was also shown to homodimerize, consistent with other SULTs that contain the consensus dimerization motif. Mutation of the dimerization motif resulted in a monomeric form of SULT4A1 that was rapidly degraded by polyubiquitination on the lysine located within the dimerization motif. These results show that SULT4A1 is widely expressed in human tissues, but mostly as a splice variant that produces a rapidly degraded protein. Dimerization protects the protein from degradation. Since many other cytosolic sulfotransferases possess the conserved lysine within the dimerization motif, homodimerization may serve, in part, to stabilize these enzymes in vivo.
    PLoS ONE 07/2014; 9(7):e101520. DOI:10.1371/journal.pone.0101520 · 3.23 Impact Factor
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    ABSTRACT: Polychlorinated biphenyls (PCBs) are persistent worldwide pollutants that are of concern due to their bioaccumulation and health effects. Metabolic oxidation of PCBs results in the formation of hydroxylated metabolites (OHPCBs). Among their biological effects, OHPCBs have been shown to alter the metabolism of endocrine hormones, including inhibition of mammalian cytosolic sulfotransferases (SULTs) that are responsible for the inactivation of thyroid hormones and phenolic steroids (i.e., hSULT1A1, hSULT1B1, and hSULT1E1). OHPCBs also interact with a human hydroxysteroid sulfotransferase that plays a role in the sulfation of endogenous alcohol-containing steroid hormones and bile acids (i.e., hSULT2A1). The objectives of our current study were to examine the effects of a series of OHPCB congeners on the activity of hSULT2A1 and to develop a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for OHPCBs as inhibitors of the enzyme. A total of 15 OHPCBs were examined, and the sulfation of 1 μM [(3)H] dehydroepiandrosterone (DHEA) was utilized as a model reaction catalyzed by the enzyme. All 15 OHPCBs inhibited the sulfation of DHEA, with IC(50) values ranging from 0.6 μM to 96 μM, and eight of these OHPCBs were also substrates for the enzyme. Comparative molecular field analysis (CoMFA) provided a predictive 3D-QSAR model with a q(2) value of 0.697 and an r(2) value of 0.949. The OHPCBs that had the highest potency as inhibitors of DHEA sulfation were those with a 3, 5-dichloro-4-hydroxy substitution pattern on the biphenyl ring system, and these congeners were also substrates for sulfation catalyzed by hSULT2A1.
    Chemical Research in Toxicology 09/2011; 24(10):1720-8. DOI:10.1021/tx200260h · 3.53 Impact Factor
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