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3'-Phosphoadenosine 5'-phosphosulfate allosterically regulates sulfotransferase turnover.

Wang T, Cook I, Leyh TS - Biochemistry (2014)

Bottom Line: The first nucleotide to bind causes closure of the cap to which it is bound and at the same time stabilizes the cap in the adjacent subunit in the open position.Cap closure sterically controls active-site access of the nucleotide and acceptor; consequently, the structural changes in the cap that occur as a function of nucleotide occupancy lead to changes in the substrate affinities and turnover of the enzyme.PAPS levels in tissues from a variety of organs suggest that the catalytic efficiency of the enzyme varies across tissues over the full 130-fold range and that efficiency is greatest in those tissues that experience the greatest xenobiotic "load".

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461-1926, United States.

ABSTRACT
Human cytosolic sulfotransferases (SULTs) regulate the activities of thousands of small molecules-metabolites, drugs, and other xenobiotics-via the transfer of the sulfuryl moiety (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyls and primary amines of acceptors. SULT1A1 is the most abundant SULT in liver and has the broadest substrate spectrum of any SULT. Here we present the discovery of a new form of SULT1A1 allosteric regulation that modulates the catalytic efficiency of the enzyme over a 130-fold dynamic range. The molecular basis of the regulation is explored in detail and is shown to be rooted in an energetic coupling between the active-site caps of adjacent subunits in the SULT1A1 dimer. The first nucleotide to bind causes closure of the cap to which it is bound and at the same time stabilizes the cap in the adjacent subunit in the open position. Binding of the second nucleotide causes both caps to open. Cap closure sterically controls active-site access of the nucleotide and acceptor; consequently, the structural changes in the cap that occur as a function of nucleotide occupancy lead to changes in the substrate affinities and turnover of the enzyme. PAPS levels in tissues from a variety of organs suggest that the catalytic efficiency of the enzyme varies across tissues over the full 130-fold range and that efficiency is greatest in those tissues that experience the greatest xenobiotic "load".

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Equilibriumbinding of PAPS to SULT1A1. (A) PAPS binding to thehigh-affinity subunit. Binding was monitored via ligand-induced changesin the intrinsic fluorescence of SULT1A1 (λex = 295nm; λem = 345 nm). Reaction conditions included SULT1A1(0.05 μM, dimer), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C. Each point is the averageof three independent determinations. The solid line through the datarepresents a least-squares fit using a model that assumes a singlebinding site per dimer. Kd = 0.37 ±0.05 μM. (B) PAPS binding stoichiometry at the high-affinitysite. The conditions were identical to those in described for panelA except that [SULT1A1] = 3.0 μM dimer (16Kd). The stoichiometry was 1.1 ± 0.2 PAPS moleculesper dimer. (C) PAPS binding at the low-affinity site. Experimentalconditions were identical to those in described for panel B. PAPSbinding is biphasic. The high- and low-affinity phases are coloredred (inset) and black, respectively. The line through the points representsa least-squares fit to the low-affinity phase using a model that assumesa single binding site per dimer. Kd =30 ± 4 μM. (D) Full-site PAPS binding stoichiometry. Thereaction conditions were identical to those described for panel Aexcept that [SULT1A1] = 475 μM dimer (16Kd for the low-affinity site). The stoichiometry was 2.1 ±0.2 PAPS molecules per dimer, or 1.1 ± 0.1 per subunit.
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fig1: Equilibriumbinding of PAPS to SULT1A1. (A) PAPS binding to thehigh-affinity subunit. Binding was monitored via ligand-induced changesin the intrinsic fluorescence of SULT1A1 (λex = 295nm; λem = 345 nm). Reaction conditions included SULT1A1(0.05 μM, dimer), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C. Each point is the averageof three independent determinations. The solid line through the datarepresents a least-squares fit using a model that assumes a singlebinding site per dimer. Kd = 0.37 ±0.05 μM. (B) PAPS binding stoichiometry at the high-affinitysite. The conditions were identical to those in described for panelA except that [SULT1A1] = 3.0 μM dimer (16Kd). The stoichiometry was 1.1 ± 0.2 PAPS moleculesper dimer. (C) PAPS binding at the low-affinity site. Experimentalconditions were identical to those in described for panel B. PAPSbinding is biphasic. The high- and low-affinity phases are coloredred (inset) and black, respectively. The line through the points representsa least-squares fit to the low-affinity phase using a model that assumesa single binding site per dimer. Kd =30 ± 4 μM. (D) Full-site PAPS binding stoichiometry. Thereaction conditions were identical to those described for panel Aexcept that [SULT1A1] = 475 μM dimer (16Kd for the low-affinity site). The stoichiometry was 2.1 ±0.2 PAPS molecules per dimer, or 1.1 ± 0.1 per subunit.

Mentions: Binding of PAPSto SULT1A1 was monitored via ligand-dependent changes in intrinsicfluorescence (λex = 290 nm; λem =345 nm). Typically, PAPS was titrated into solutions containing SULT1A1,MgCl2 (5.0 mM), and NaPO4 (50 mM) at pH 7.2and 25 ± 2 °C. The PAPS concentrations used in the titrationdepicted in Figure 1D were high enough to causeinner-filter effects. Consequently, λex was shiftedto 297 nm to lower the PAPS absorbance (ε297 = 0.43mM–1 cm–1). Despite the loweredabsorbance, inner-filter effects were detected at ≥60 μMPAPS. To correct for these effects, control titrations in which AMP(which does not bind SULT1A1) was substituted for PAPS were performedand the PAPS titration data were corrected accordingly. All titrationswere performed in triplicate, and the averaged data were least-squaresfit using a model that assumes a single binding site per dimer.12


3'-Phosphoadenosine 5'-phosphosulfate allosterically regulates sulfotransferase turnover.

Wang T, Cook I, Leyh TS - Biochemistry (2014)

Equilibriumbinding of PAPS to SULT1A1. (A) PAPS binding to thehigh-affinity subunit. Binding was monitored via ligand-induced changesin the intrinsic fluorescence of SULT1A1 (λex = 295nm; λem = 345 nm). Reaction conditions included SULT1A1(0.05 μM, dimer), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C. Each point is the averageof three independent determinations. The solid line through the datarepresents a least-squares fit using a model that assumes a singlebinding site per dimer. Kd = 0.37 ±0.05 μM. (B) PAPS binding stoichiometry at the high-affinitysite. The conditions were identical to those in described for panelA except that [SULT1A1] = 3.0 μM dimer (16Kd). The stoichiometry was 1.1 ± 0.2 PAPS moleculesper dimer. (C) PAPS binding at the low-affinity site. Experimentalconditions were identical to those in described for panel B. PAPSbinding is biphasic. The high- and low-affinity phases are coloredred (inset) and black, respectively. The line through the points representsa least-squares fit to the low-affinity phase using a model that assumesa single binding site per dimer. Kd =30 ± 4 μM. (D) Full-site PAPS binding stoichiometry. Thereaction conditions were identical to those described for panel Aexcept that [SULT1A1] = 475 μM dimer (16Kd for the low-affinity site). The stoichiometry was 2.1 ±0.2 PAPS molecules per dimer, or 1.1 ± 0.1 per subunit.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230322&req=5

fig1: Equilibriumbinding of PAPS to SULT1A1. (A) PAPS binding to thehigh-affinity subunit. Binding was monitored via ligand-induced changesin the intrinsic fluorescence of SULT1A1 (λex = 295nm; λem = 345 nm). Reaction conditions included SULT1A1(0.05 μM, dimer), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C. Each point is the averageof three independent determinations. The solid line through the datarepresents a least-squares fit using a model that assumes a singlebinding site per dimer. Kd = 0.37 ±0.05 μM. (B) PAPS binding stoichiometry at the high-affinitysite. The conditions were identical to those in described for panelA except that [SULT1A1] = 3.0 μM dimer (16Kd). The stoichiometry was 1.1 ± 0.2 PAPS moleculesper dimer. (C) PAPS binding at the low-affinity site. Experimentalconditions were identical to those in described for panel B. PAPSbinding is biphasic. The high- and low-affinity phases are coloredred (inset) and black, respectively. The line through the points representsa least-squares fit to the low-affinity phase using a model that assumesa single binding site per dimer. Kd =30 ± 4 μM. (D) Full-site PAPS binding stoichiometry. Thereaction conditions were identical to those described for panel Aexcept that [SULT1A1] = 475 μM dimer (16Kd for the low-affinity site). The stoichiometry was 2.1 ±0.2 PAPS molecules per dimer, or 1.1 ± 0.1 per subunit.
Mentions: Binding of PAPSto SULT1A1 was monitored via ligand-dependent changes in intrinsicfluorescence (λex = 290 nm; λem =345 nm). Typically, PAPS was titrated into solutions containing SULT1A1,MgCl2 (5.0 mM), and NaPO4 (50 mM) at pH 7.2and 25 ± 2 °C. The PAPS concentrations used in the titrationdepicted in Figure 1D were high enough to causeinner-filter effects. Consequently, λex was shiftedto 297 nm to lower the PAPS absorbance (ε297 = 0.43mM–1 cm–1). Despite the loweredabsorbance, inner-filter effects were detected at ≥60 μMPAPS. To correct for these effects, control titrations in which AMP(which does not bind SULT1A1) was substituted for PAPS were performedand the PAPS titration data were corrected accordingly. All titrationswere performed in triplicate, and the averaged data were least-squaresfit using a model that assumes a single binding site per dimer.12

Bottom Line: The first nucleotide to bind causes closure of the cap to which it is bound and at the same time stabilizes the cap in the adjacent subunit in the open position.Cap closure sterically controls active-site access of the nucleotide and acceptor; consequently, the structural changes in the cap that occur as a function of nucleotide occupancy lead to changes in the substrate affinities and turnover of the enzyme.PAPS levels in tissues from a variety of organs suggest that the catalytic efficiency of the enzyme varies across tissues over the full 130-fold range and that efficiency is greatest in those tissues that experience the greatest xenobiotic "load".

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461-1926, United States.

ABSTRACT
Human cytosolic sulfotransferases (SULTs) regulate the activities of thousands of small molecules-metabolites, drugs, and other xenobiotics-via the transfer of the sulfuryl moiety (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyls and primary amines of acceptors. SULT1A1 is the most abundant SULT in liver and has the broadest substrate spectrum of any SULT. Here we present the discovery of a new form of SULT1A1 allosteric regulation that modulates the catalytic efficiency of the enzyme over a 130-fold dynamic range. The molecular basis of the regulation is explored in detail and is shown to be rooted in an energetic coupling between the active-site caps of adjacent subunits in the SULT1A1 dimer. The first nucleotide to bind causes closure of the cap to which it is bound and at the same time stabilizes the cap in the adjacent subunit in the open position. Binding of the second nucleotide causes both caps to open. Cap closure sterically controls active-site access of the nucleotide and acceptor; consequently, the structural changes in the cap that occur as a function of nucleotide occupancy lead to changes in the substrate affinities and turnover of the enzyme. PAPS levels in tissues from a variety of organs suggest that the catalytic efficiency of the enzyme varies across tissues over the full 130-fold range and that efficiency is greatest in those tissues that experience the greatest xenobiotic "load".

Show MeSH
Related in: MedlinePlus