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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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Binding of TAM to E and E·(PAP)2. (A) TAM bindingto E. Binding was monitored via changes in SULT1A1 intrinsic fluorescence(λex = 290 nm; λem = 345 nm). Reactionconditions included SULT1A1 (0.10 μM, dimer), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C.Each point is the average of three independent determinations. Thecurve is the behavior predicted by a best fit model that assumes asingle binding site per dimer. Kd = 0.67± 0.04. (B) TAM binding to E(PAP)2. Conditions anddata analysis were identical to those described for panel A exceptPAP = 0.50 mM (17Kd for PAPS binding atits low-affinity site). The Kd for TAMbinding is 0.68 ± 0.12 μM. (C) Stoichiometry of bindingof TAM to E and E·(PAP)2. Conditions were identicalto those described for panels A and B except that [SULT1A1] = 10 μM(dimer). Binding to E and E·(PAP)2 is shown with filledand empty circles, respectively. The stoichiometries are 2.0 ±0.1 TAM bound per SULT1A1 dimer.
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fig3: Binding of TAM to E and E·(PAP)2. (A) TAM bindingto E. Binding was monitored via changes in SULT1A1 intrinsic fluorescence(λex = 290 nm; λem = 345 nm). Reactionconditions included SULT1A1 (0.10 μM, dimer), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C.Each point is the average of three independent determinations. Thecurve is the behavior predicted by a best fit model that assumes asingle binding site per dimer. Kd = 0.67± 0.04. (B) TAM binding to E(PAP)2. Conditions anddata analysis were identical to those described for panel A exceptPAP = 0.50 mM (17Kd for PAPS binding atits low-affinity site). The Kd for TAMbinding is 0.68 ± 0.12 μM. (C) Stoichiometry of bindingof TAM to E and E·(PAP)2. Conditions were identicalto those described for panels A and B except that [SULT1A1] = 10 μM(dimer). Binding to E and E·(PAP)2 is shown with filledand empty circles, respectively. The stoichiometries are 2.0 ±0.1 TAM bound per SULT1A1 dimer.

Mentions: Theaffinities and stoichiometries of binding of TAM to SULT1A1 at 0 and0.50 mM PAP (which is sufficient to saturate both nucleotide pockets)were determined by fluorescence titration (Figure 3A–C). The results, compiled in Table 2, reveal that the TAM affinities for E·PAP and E·(PAP)2 are virtually identical (0.67 ± 0.08 and 0.65 ±0.07 μM, respectively) and that each subunit of the dimer bindsone acceptor. In contrast, when the PAP concentration favors the singlynucleotide-bound dimer, TAM binding is biphasic (Figure 4A,B). At 6.0 μM PAP, the distribution of forms is biasedtoward the E·PAP complex [E·PAP, 79%; E·(PAP)2, 16%; E, 5.0%] and the affinities of the phases (0.67 ± 0.03and 13 ± 2 μM) strongly suggest that the cap of one subunitis open while that of the other is closed. To confirm that each dimercontains a single high-affinity site, a “stoichiometry”titration was performed at a dimer concentration of 9.0 μM [i.e.,13Kd TAM, 24Kd PAP(high affinity), and 0.3Kd PAP(low affinity)]. To maximize the concentrationof singly bound species, the nucleotide concentration was set equalto that of the dimer, 9.0 μM. Under this condition, the distributionof forms is as follows: E·PAP, 77%; E·(PAP)2,3.8%; E, 19%. Both high- and low-affinity sites are observed in thetitration, and the inset reveals that each dimer contains a singlehigh-affinity TAM binding site.


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

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

Binding of TAM to E and E·(PAP)2. (A) TAM bindingto E. Binding was monitored via changes in SULT1A1 intrinsic fluorescence(λex = 290 nm; λem = 345 nm). Reactionconditions included SULT1A1 (0.10 μM, dimer), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C.Each point is the average of three independent determinations. Thecurve is the behavior predicted by a best fit model that assumes asingle binding site per dimer. Kd = 0.67± 0.04. (B) TAM binding to E(PAP)2. Conditions anddata analysis were identical to those described for panel A exceptPAP = 0.50 mM (17Kd for PAPS binding atits low-affinity site). The Kd for TAMbinding is 0.68 ± 0.12 μM. (C) Stoichiometry of bindingof TAM to E and E·(PAP)2. Conditions were identicalto those described for panels A and B except that [SULT1A1] = 10 μM(dimer). Binding to E and E·(PAP)2 is shown with filledand empty circles, respectively. The stoichiometries are 2.0 ±0.1 TAM bound per SULT1A1 dimer.
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Related In: Results  -  Collection

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fig3: Binding of TAM to E and E·(PAP)2. (A) TAM bindingto E. Binding was monitored via changes in SULT1A1 intrinsic fluorescence(λex = 290 nm; λem = 345 nm). Reactionconditions included SULT1A1 (0.10 μM, dimer), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, and 25 ± 2 °C.Each point is the average of three independent determinations. Thecurve is the behavior predicted by a best fit model that assumes asingle binding site per dimer. Kd = 0.67± 0.04. (B) TAM binding to E(PAP)2. Conditions anddata analysis were identical to those described for panel A exceptPAP = 0.50 mM (17Kd for PAPS binding atits low-affinity site). The Kd for TAMbinding is 0.68 ± 0.12 μM. (C) Stoichiometry of bindingof TAM to E and E·(PAP)2. Conditions were identicalto those described for panels A and B except that [SULT1A1] = 10 μM(dimer). Binding to E and E·(PAP)2 is shown with filledand empty circles, respectively. The stoichiometries are 2.0 ±0.1 TAM bound per SULT1A1 dimer.
Mentions: Theaffinities and stoichiometries of binding of TAM to SULT1A1 at 0 and0.50 mM PAP (which is sufficient to saturate both nucleotide pockets)were determined by fluorescence titration (Figure 3A–C). The results, compiled in Table 2, reveal that the TAM affinities for E·PAP and E·(PAP)2 are virtually identical (0.67 ± 0.08 and 0.65 ±0.07 μM, respectively) and that each subunit of the dimer bindsone acceptor. In contrast, when the PAP concentration favors the singlynucleotide-bound dimer, TAM binding is biphasic (Figure 4A,B). At 6.0 μM PAP, the distribution of forms is biasedtoward the E·PAP complex [E·PAP, 79%; E·(PAP)2, 16%; E, 5.0%] and the affinities of the phases (0.67 ± 0.03and 13 ± 2 μM) strongly suggest that the cap of one subunitis open while that of the other is closed. To confirm that each dimercontains a single high-affinity site, a “stoichiometry”titration was performed at a dimer concentration of 9.0 μM [i.e.,13Kd TAM, 24Kd PAP(high affinity), and 0.3Kd PAP(low affinity)]. To maximize the concentrationof singly bound species, the nucleotide concentration was set equalto that of the dimer, 9.0 μM. Under this condition, the distributionof forms is as follows: E·PAP, 77%; E·(PAP)2,3.8%; E, 19%. Both high- and low-affinity sites are observed in thetitration, and the inset reveals that each dimer contains a singlehigh-affinity TAM binding site.

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