Article

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.

0 Followers
 · 
283 Views
  • Source
    [Show abstract] [Hide abstract]
    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 · 4.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The human cytosolic sulfotransferase hSULT2A1 catalyzes the sulfation of a broad range of xenobiotics as well as endogenous hydroxysteroids and bile acids. Reversible modulation of the catalytic activity of this enzyme could play important roles in its physiological functions. While other mammalian sulfotransferases are known to be reversibly altered by changes in their redox environment, this has not been previously shown for hSULT2A1. We have examined the hypothesis that the formation of disulfide bonds in hSULT2A1 can reversibly regulate the catalytic function of the enzyme. Three thiol-oxidants were utilized as model compounds to investigate their effects on homogeneous preparations of hSULT2A1: glutathione disulfide (GSSG), 5,5(')-dithiobis(2-nitrobenzoic acid) (DTNB), and 1,1(')-azobis(N,N-dimethylformamide) (Diamide). Examination of the effects of disulfide bond formation with these agents indicated that the activity of the enzyme is reversibly altered. Studies on the kinetics of the hSULT2A1-catalyzed sulfation of dehydroepiandrosterone (DHEA) showed effects of disulfide bond formation on the substrate inhibition characteristics of the enzyme. The effects of these agents on the binding of substrates and products, LC-MS identification of the disulfides formed, and structural modeling of the modified enzyme were examined. Our results indicated that conformational changes at cysteines near the PAPS/PAP binding site affect binding of both the nucleotide and DHEA to the enzyme, with the specific effects dependent upon the structure of the resulting disulfide. Thus, the formation of disulfide bonds in hSULT2A1 is a potentially important reversible mechanism for alterations in the rates of sulfation of both endogenous and xenobiotic substrates.
    Drug metabolism and disposition: the biological fate of chemicals 02/2013; 41(5). DOI:10.1124/dmd.112.050534 · 3.33 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Abstract The SULT2b gene family consists of a single gene capable of generating two functional transcripts utilizing different transcriptional start sites in the first exon. This results in the translation of two separate proteins, SULT2B1a and SULT2B1b, with different amino-terminal peptides and approximately 95% identical sequences. The second distinguishing feature of the SULT2B isoforms is the proline/serine-rich carboxy-terminal sequence. To date, presence of the SULT2B gene appears limited to mammals and there is also only limited conservation of structure or sequence of the carboxy-terminal peptide. Although both SULT2B1 messages are present in human tissues, to date, only the SULT2B1b protein has been detected in the tissues investigated. In contrast, selective expression of SULT2B1a has been detected in rodent brain, whereas SULT2B1b was expressed in skin and intestine. Characterization of the SULT2B1 isoforms has been limited by the inability to isolate reliably active SULT2B1b from tissues or cells. SULT2B1 cDNAs can be expressed in Escherichia coli and the expressed active enzymes show selectivity for sulfation of 3β-hydroxysteroids. SULT2B1b due to the binding properties of the amino-terminal peptides also shows high cholesterol sulfation activity. Although human SULT2B1b displays significant substrate cross-reactivity with SULT2A1, the isoforms have different tissue expression patterns. Human SULT2B1b also shows nuclear localization in selected tissues that appears related to serine phosphorylation of the carboxy-terminal peptide. Overall, the understanding of the properties and function of the SULT2B1 isoforms is limited and the structural variability of the unique amino- and carboxy-sequences suggests significant species differences that need to be investigated.
    Drug Metabolism Reviews 09/2013; DOI:10.3109/03602532.2013.835609 · 6.29 Impact Factor
Show more

Preview

Download
2 Downloads
Available from