Redox Control of Aryl Sulfotransferase Specificity

doi:10.1006/abbi.2000.2020

Archives of Biochemistry and Biophysics

Volume 382, Issue 1, 1 October 2000, Pages 95-104

https://doi.org/10.1006/abbi.2000.2020 Get rights and content

Abstract

Aryl sulfotransferase IV from rat liver has the very broad substrate range that is characteristic of the enzymes of detoxication. With the conventional assay substrates, 4-nitrophenol and PAPS, sulfation was considered optimal at pH 5.5 whereas the enzyme in the physiological pH range was curiously ineffective. These properties would seem to preclude a physiological function for this cytosolic enzyme. Partial oxidation of the enzyme, however, results not only in a substantial increase in the rate of sulfation of 4-nitrophenol at physiological pH but also in a shift of the pH optimum to this range and radically altered overall substrate specificity. The mechanism for this dependence on redox environment involves oxidation at Cys66, a process previously shown to occur by formation of a mixed disulfide with glutathione or by the formation of an internal disulfide with Cys232. Oxidation at Cys66 acts only as a molecular redox switch and is not directly part of the catalytic mechanism. Underlying the activation process is a change in the nature of the ternary complex formed between enzyme, phenol, and the reaction product, adenosine 3′,5′-bisphosphate. The reduced enzyme gives rise to an inhibitory, dead-end ternary complex, the stability of which is dictated by the ionization of the specific phenol substrate. Ternary complex formation impedes the binding of PAPS that is necessary to initiate a further round of the reaction and is manifest as profound, substrate-dependent inhibition. In contrast, the ternary complex formed when the enzyme is in the partially oxidized state allows binding of PAPS and the unhindered completion of the reaction cycle.

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Sulfotransferases catalyze the formation of sulfuric acid esters, most often referred to as sulfates, from a wide range of xenobiotics and their metabolites, as well as various endogenous neurotransmitters, hormones, bile acids, carbohydrates, and proteins. This article is focused on the cytosolic sulfotransferases, a superfamily of enzymes that, depending on the specific substrates and products of the sulfation reaction, are responsible for either detoxication or metabolic activation of a wide range of drugs, toxins, carcinogens, environmental chemicals, and other xenobiotics. An overview of current aspects of the nomenclature, gene organization, regulation of expression, enzyme structures and catalytic mechanisms, specificities for substrates and inhibitors, and roles in toxicology is provided for these cytosolic sulfotransferases.
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Cellular redox signaling is important in diverse physiological and pathological processes. The activity of rat phenol sulfotransferase (rSULT1A1), which is important for the metabolism of hormone and drug, is subjected to redox regulation. Two cysteines, Cys232 and Cys66, nanometer away from each other and from the enzyme active site were proposed to form disulfide bond to regulate the activity of rSULT1A1. A nano switch, composed of a flexible loop from amino acid residues 59–70, explained how this long distance interaction between two cysteines can be achieved. The enzyme properties were investigated through site-directed muatagnesis, circular dichroism, enzyme kinetics and homologous modeling of the rSULT1A1 structures. We proposed that the formation of disulfide bond between Cys232 and Cys66 induced conformational changes of sulfotransferase, then in turn affected its nucleotide binding and enzyme activity. This discovery was extended to understand the possible redox regulation of other sulfotransferases from different organisms. The redox switch can be created in other redox-insensitive sulfotransferases, such as human phenol sulfotransferase (hSULT1A1) and human alcohol sulfotransferase (hSULT2A1), to produce mutant enzymes with redox regulation capacity. This study strongly suggested that redox regulation of drug and hormone metabolism can be significantly varied even though the sequence and structure of SULT1A1 of human and rat have a high degree of homology.
Substrate inhibition in human hydroxysteroid sulfotransferase SULT2A1: Studies on the formation of catalytically non-productive enzyme complexes
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Citation Excerpt :
Studies on the catalytic mechanism of cytosolic sulfotransferases have been primarily focused on the SULT1 family (previously known as either as phenol or aryl sulfotransferases). Kinetic and crystallographic studies on these enzymes have elucidated several general principles regarding the chemical mechanism of sulfuryl transfer from PAPS to an acceptor substrate [1,24–31], that, based on three-dimensional structural homologies, may be applicable to members of other SULT families. Details of the kinetic mechanisms for individual SULTs, however, continue to provide information on the substrate-dependent differences in sulfation reactions that define the specificities and functions of these enzymes.
The cytosolic sulfotransferase hSULT2A1 is the major hydroxysteroid (alcohol) sulfotransferase in human liver, and it catalyzes the 3′-phosphoadenosine-5′-phosphosulfate (PAPS)-dependent sulfation of various endogenous hydroxysteroids as well as many xenobiotics that contain alcohol and phenol functional groups. The hSULT2A1 often displays substrate inhibition, and we have hypothesized that a key element in this response to increasing substrate concentration is the formation of non-productive ternary dead-end enzyme complexes involving the nucleotide product, adenosine 3′,5′-diphosphate (PAP). One of these substrates for hSULT2A1 is dehydroepiandrosterone (DHEA), a major circulating steroid hormone in humans that serves as precursor to both androgens and estrogens. We have utilized DHEA in both initial velocity studies and equilibrium binding experiments in order to evaluate the potential role of ternary complexes in substrate inhibition of the enzyme. Our results indicate that hSULT2A1 forms non-productive ternary complexes that involve either DHEA or dehydroepiandrosterone sulfate, and the formation of these ternary complexes displays negative cooperativity in the binding of DHEA.
Physicochemical properties of hydroxylated polychlorinated biphenyls aid in predicting their interactions with rat sulfotransferase 1A1 (rSULT1A1)
2011, Chemico-Biological Interactions
Citation Excerpt :
This substrate inhibition is due to the formation of dead end complexes between the substrate, enzyme and the co-product of sulfation, adenosine-3′,5′-diphosphate (PAP) [26,27]. The release of PAP from these dead-end complexes becomes a rate-limiting component of the rSULT1A1-catalyzed reaction [27], and changes in the conformation at the PAP/PAPS-binding site of the enzyme may affect catalytic function and inhibition through facilitation of PAP-release [2,27]. Thus the present studies are focused on molecular characteristics of OHPCB-substrates that influence the rate of reaction, the use of calculated estimates of physicochemical parameters in the prediction of interactions of OHPCBs as substrates and inhibitors, and the potential role of the redox status of the enzyme in determining its binding interactions with OHPCBs and PAP.
Hydroxylated metabolites of polychlorinated biphenyls (OHPCBs) interact with rat sulfotransferase 1A1 (rSULT1A1) as substrates and inhibitors. Previous studies have shown that there are complex and incompletely understood structure–activity relationships governing the interaction of rSULT1A1 with these molecules. Furthermore, modification of the enzyme with glutathione disulfide (GSSG) results in the conversion of some OHPCBs from inhibitors to substrates. We have now examined estimated values for the acid-dissociation constant (K_a) and the octanol–water distribution coefficient (D), as well as experimentally determined dissociation constants for enzyme complexes, to assist in the prediction of interactions of OHPCBs with rSULT1A1. Under reducing conditions, initial velocities for rSULT1A1-catalyzed sulfation exhibited a positive correlation with pK_a and a negative correlation with log D of the OHPCBs. IC₅₀ values of inhibitory OHPCBs decreased with decreasing pK_a values for both the glutathione (GSH)-pretreated and GSSG-pretreated forms of rSULT1A1. Comparison of GSH- and GSSG-pretreated forms of rSULT1A1 with respect to binding of OHPCB in the presence and absence of adenosine 3′,5′-diphosphate (PAP) revealed that the dissociation constants with the two redox states of the enzyme were similar for each OHPCB. Thus, pK_a and log D values are useful in predicting the binding of OHPCBs to the two redox forms of rSULT1A1 as well as the rates of sulfation of those OHPCBs that are substrates. However, the differences in substrate specificity for OHPCBs that are seen with changes in redox status of the enzyme are not directly related to specific structural effects of individual OHPCBs within inhibitory enzyme–PAP–OHPCB complexes.
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Sulfotransferases catalyze the formation of sulfuric acid esters, most often referred to as sulfates, from a wide range of xenobiotics and their metabolites, as well as various endogenous neurotransmitters, hormones, bile acids, carbohydrates, and proteins. This chapter is focused on the cytosolic sulfotransferases, a superfamily of enzymes that, depending on the specific substrates and products of the sulfation reaction, are responsible for either detoxication or metabolic activation of a wide range of drugs, toxins, carcinogens, environmental chemicals, and other xenobiotics. An overview of current aspects of the nomenclature, gene organization, regulation of expression, enzyme structures and catalytic mechanisms, specificities for substrates and inhibitors, and roles in toxicology is provided for these cytosolic sulfotransferases.
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¹: Present address: Novartis Institute for Medical Sciences, 5 Gower Place, London WC1E 6BN, UK.

²: To whom correspondence should be addressed at the National Institutes of Health, Building 8, Room 311, Bethesda, MD 20892. Fax: (301) 480-0040. E-mail: [email protected].

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Regular ArticleRedox Control of Aryl Sulfotransferase Specificity

Abstract

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

Arch. Biochem. Biophys.

Protein Expr. Purif.

J. Biol. Chem.

J. Biol. Chem.

Protein Expr. Purif.

Chem. Biol. Interact.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Chem. Biol. Interact.

J. Biol. Chem.

Methods Enzymol.

Regular Article
Redox Control of Aryl Sulfotransferase Specificity