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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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SUTL1A1 turnover as afunction of PAPS occupancy. A plot of SULT1Aturnover vs [PAPS] is biphasic. The first and second phases correspondto saturation of the high- and low-affinity PAPS binding sites, respectively.The reaction was monitored via 1-HPS fluorescence (λex = 320 nm; λem = 380 nm). The conditions includedSULT1A1 (1.0 nM, dimer), 1-HP (160 μM, 20Kd), MgCl2 (5.0 mM), NaPO4 (50 mM), pH7.2, and 25 ± 2.0 °C. The asterisks indicate the PAPS concentrations(0.20 and 2.0 μM) used in initial rate studies to obtain Michaelisparameters for the E·PAP and E·(PAPS)2 forms.
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fig5: SUTL1A1 turnover as afunction of PAPS occupancy. A plot of SULT1Aturnover vs [PAPS] is biphasic. The first and second phases correspondto saturation of the high- and low-affinity PAPS binding sites, respectively.The reaction was monitored via 1-HPS fluorescence (λex = 320 nm; λem = 380 nm). The conditions includedSULT1A1 (1.0 nM, dimer), 1-HP (160 μM, 20Kd), MgCl2 (5.0 mM), NaPO4 (50 mM), pH7.2, and 25 ± 2.0 °C. The asterisks indicate the PAPS concentrations(0.20 and 2.0 μM) used in initial rate studies to obtain Michaelisparameters for the E·PAP and E·(PAPS)2 forms.

Mentions: When SULT1A1 turnoveris plotted versus PAPS concentration at a saturating 1-HP concentration(Figure 5), distinct low- and high-PAPS affinityphases are observed. To quantitate the differences in initial ratebehavior of the singly and doubly occupied enzyme, initial rate experimentswere performed at PAPS concentrations fixed in the plateau regionof each phase (see asterisks in Figure 5).Table 3 lists the initial rate parameters associatedwith 1-HP sulfonation and the PAPS concentrations at which they weredetermined. As expected for a small acceptor, Km for 1-HP is not affected by PAPS occupancy; however, kcat increases nearly 8-fold. If the increasedrate of turnover were due solely to PAPS saturation at the secondsite, a 2-fold increase would have occurred. Thus, as the caps areopened at both sites, each subunit turns over 4 times more quicklythan the subunit in the singly occupied enzyme.


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

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

SUTL1A1 turnover as afunction of PAPS occupancy. A plot of SULT1Aturnover vs [PAPS] is biphasic. The first and second phases correspondto saturation of the high- and low-affinity PAPS binding sites, respectively.The reaction was monitored via 1-HPS fluorescence (λex = 320 nm; λem = 380 nm). The conditions includedSULT1A1 (1.0 nM, dimer), 1-HP (160 μM, 20Kd), MgCl2 (5.0 mM), NaPO4 (50 mM), pH7.2, and 25 ± 2.0 °C. The asterisks indicate the PAPS concentrations(0.20 and 2.0 μM) used in initial rate studies to obtain Michaelisparameters for the E·PAP and E·(PAPS)2 forms.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230322&req=5

fig5: SUTL1A1 turnover as afunction of PAPS occupancy. A plot of SULT1Aturnover vs [PAPS] is biphasic. The first and second phases correspondto saturation of the high- and low-affinity PAPS binding sites, respectively.The reaction was monitored via 1-HPS fluorescence (λex = 320 nm; λem = 380 nm). The conditions includedSULT1A1 (1.0 nM, dimer), 1-HP (160 μM, 20Kd), MgCl2 (5.0 mM), NaPO4 (50 mM), pH7.2, and 25 ± 2.0 °C. The asterisks indicate the PAPS concentrations(0.20 and 2.0 μM) used in initial rate studies to obtain Michaelisparameters for the E·PAP and E·(PAPS)2 forms.
Mentions: When SULT1A1 turnoveris plotted versus PAPS concentration at a saturating 1-HP concentration(Figure 5), distinct low- and high-PAPS affinityphases are observed. To quantitate the differences in initial ratebehavior of the singly and doubly occupied enzyme, initial rate experimentswere performed at PAPS concentrations fixed in the plateau regionof each phase (see asterisks in Figure 5).Table 3 lists the initial rate parameters associatedwith 1-HP sulfonation and the PAPS concentrations at which they weredetermined. As expected for a small acceptor, Km for 1-HP is not affected by PAPS occupancy; however, kcat increases nearly 8-fold. If the increasedrate of turnover were due solely to PAPS saturation at the secondsite, a 2-fold increase would have occurred. Thus, as the caps areopened at both sites, each subunit turns over 4 times more quicklythan the subunit in the singly occupied enzyme.

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