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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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Pre-steady-state binding of PAPS to SULT1A1. (A) Composite kobs vs [PAPS] plot. Two well-isolated bindingphases are observed. Binding was monitored via changes in SULT1A1intrinsic fluorescence (λex = 290 nm; λem ≥ 330 nm). kobs valuesare the average of three independent determinations. Reaction conditionsincluded SULT1A1 (0.050 μM, dimer), MgCl2 (5.0 mM),NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C. Red dotsindicate the kobs values predicted usingthe kon and koff values obtained from the experiments associated with panels B andC. (B) kobs vs [PAPS] for the high-affinitysubunit. Reaction conditions were identical to those described forpanel A except that [SULT1A1] = 0.030 μM (dimer). kon = 2.0 ± 0.2 μM–1 s–1; koff = 0.70 ± 0.02s–1. (C) kobs vs [PAPS]for the low-affinity subunit. Reaction conditions were identical tothose described for panel A except the SULT1A1 (2.0 μM, dimer)was equilibrated with PAPS [8.0 μM, 26Kd(high affinity), 0.27Kd(low affinity)] before being mixed with PAPS at higher concentrations (20–80μM). kon = 0.96 ± 0.01 μMs–1; koff = 29 ±1 s–1. All reactions were pseudo-first-order inPAPS concentration.
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fig2: Pre-steady-state binding of PAPS to SULT1A1. (A) Composite kobs vs [PAPS] plot. Two well-isolated bindingphases are observed. Binding was monitored via changes in SULT1A1intrinsic fluorescence (λex = 290 nm; λem ≥ 330 nm). kobs valuesare the average of three independent determinations. Reaction conditionsincluded SULT1A1 (0.050 μM, dimer), MgCl2 (5.0 mM),NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C. Red dotsindicate the kobs values predicted usingthe kon and koff values obtained from the experiments associated with panels B andC. (B) kobs vs [PAPS] for the high-affinitysubunit. Reaction conditions were identical to those described forpanel A except that [SULT1A1] = 0.030 μM (dimer). kon = 2.0 ± 0.2 μM–1 s–1; koff = 0.70 ± 0.02s–1. (C) kobs vs [PAPS]for the low-affinity subunit. Reaction conditions were identical tothose described for panel A except the SULT1A1 (2.0 μM, dimer)was equilibrated with PAPS [8.0 μM, 26Kd(high affinity), 0.27Kd(low affinity)] before being mixed with PAPS at higher concentrations (20–80μM). kon = 0.96 ± 0.01 μMs–1; koff = 29 ±1 s–1. All reactions were pseudo-first-order inPAPS concentration.

Mentions: To test the coupled cap model and distinguishbetween the adjacent cap open and closed mechanisms, the on and offrate constants for binding of PAPS to SULT1A1 were determined overa range of PAPS concentrations that probe binding to the tight andweak sites. PAPS binding was monitored via binding-induced changesin the intrinsic fluorescence of SULT1A1 (Figure S1 of the Supporting Information shows a typical PAPS bindingreaction). The reactions were pseudo-first-order in PAPS concentration,and kobs values were obtained by fittingprogress curves to a single-exponential model. The results are compiledin the kobs versus PAPS concentrationplot presented in Figure 2A, which shows twodistinct linear phases indicative of two experimentally separablebinding sites.


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

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

Pre-steady-state binding of PAPS to SULT1A1. (A) Composite kobs vs [PAPS] plot. Two well-isolated bindingphases are observed. Binding was monitored via changes in SULT1A1intrinsic fluorescence (λex = 290 nm; λem ≥ 330 nm). kobs valuesare the average of three independent determinations. Reaction conditionsincluded SULT1A1 (0.050 μM, dimer), MgCl2 (5.0 mM),NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C. Red dotsindicate the kobs values predicted usingthe kon and koff values obtained from the experiments associated with panels B andC. (B) kobs vs [PAPS] for the high-affinitysubunit. Reaction conditions were identical to those described forpanel A except that [SULT1A1] = 0.030 μM (dimer). kon = 2.0 ± 0.2 μM–1 s–1; koff = 0.70 ± 0.02s–1. (C) kobs vs [PAPS]for the low-affinity subunit. Reaction conditions were identical tothose described for panel A except the SULT1A1 (2.0 μM, dimer)was equilibrated with PAPS [8.0 μM, 26Kd(high affinity), 0.27Kd(low affinity)] before being mixed with PAPS at higher concentrations (20–80μM). kon = 0.96 ± 0.01 μMs–1; koff = 29 ±1 s–1. All reactions were pseudo-first-order inPAPS concentration.
© Copyright Policy
Related In: Results  -  Collection

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fig2: Pre-steady-state binding of PAPS to SULT1A1. (A) Composite kobs vs [PAPS] plot. Two well-isolated bindingphases are observed. Binding was monitored via changes in SULT1A1intrinsic fluorescence (λex = 290 nm; λem ≥ 330 nm). kobs valuesare the average of three independent determinations. Reaction conditionsincluded SULT1A1 (0.050 μM, dimer), MgCl2 (5.0 mM),NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C. Red dotsindicate the kobs values predicted usingthe kon and koff values obtained from the experiments associated with panels B andC. (B) kobs vs [PAPS] for the high-affinitysubunit. Reaction conditions were identical to those described forpanel A except that [SULT1A1] = 0.030 μM (dimer). kon = 2.0 ± 0.2 μM–1 s–1; koff = 0.70 ± 0.02s–1. (C) kobs vs [PAPS]for the low-affinity subunit. Reaction conditions were identical tothose described for panel A except the SULT1A1 (2.0 μM, dimer)was equilibrated with PAPS [8.0 μM, 26Kd(high affinity), 0.27Kd(low affinity)] before being mixed with PAPS at higher concentrations (20–80μM). kon = 0.96 ± 0.01 μMs–1; koff = 29 ±1 s–1. All reactions were pseudo-first-order inPAPS concentration.
Mentions: To test the coupled cap model and distinguishbetween the adjacent cap open and closed mechanisms, the on and offrate constants for binding of PAPS to SULT1A1 were determined overa range of PAPS concentrations that probe binding to the tight andweak sites. PAPS binding was monitored via binding-induced changesin the intrinsic fluorescence of SULT1A1 (Figure S1 of the Supporting Information shows a typical PAPS bindingreaction). The reactions were pseudo-first-order in PAPS concentration,and kobs values were obtained by fittingprogress curves to a single-exponential model. The results are compiledin the kobs versus PAPS concentrationplot presented in Figure 2A, which shows twodistinct linear phases indicative of two experimentally separablebinding sites.

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