Inactivation of Steroid Sulfatase by an Active Site-Directed Inhibitor, Estrone-3-O-Sulfamate

A PUROHIT, G J Williams, N M HOWARTH, B V L Potter, M J REED

Research output: Contribution to journalArticle

Abstract

Steroid sulfatases are responsible for the hydrolysis of 3 beta-hydroxy steroid sulfates, such as cholesterol and pregnenolone sulfate, and have an important role in regulating the synthesis of estrogenic steroids, from estrone sulfate and dehydroepiandrosterone sulfate, in endocrine-dependent tumors. Although little is known about the mechanism by which the sulfate group is removed from a steroid nucleus, an active site-directed sulfatase inhibitor has been developed. This inhibitor, estrone-3-O-sulfamate (EMATE), was synthesized by treating the sodium salt of estrone with sulfamoyl chloride. This compound inhibited not only estrone sulfatase but also dehydroepiandrosterone sulfatase activity in placental microsomes and in intact MCF-7 breast cancer cells. Pretreatment of MCF-7 cells or placental microsomes with EMATE, followed by extensive washing or dialysis indicated irreversible inhibition. This was confirmed by showing that EMATE inhibited estrone sulfatase activity in placental microsomes in a time-, concentration-, and pH-dependent manner. The enzyme is protected from inactivation by estrone sulfate, which is also consistent with active site-directed inhibition. EMATE is proposed to inactivate estrone sulfatase by irreversible sulfamoylation of the enzyme. Maximum enzyme activity was detected at pH 8.6, and the maximum rate of enzyme inactivation by EMATE also occurred at this pH. The pK(a) values of the enzymatic reaction and pK(a) of inactivation were 7.2 and 9.8, providing evidence that two active site residues are being modified by EMATE. As the phenolic pK(a) of tyrosine (9.7) and the pK(a) of histidine (6.8) are similar to the pK(a) values of inactivation, these amino acid residues may play a role in the catalytic mechanism. The development of an active site-directed irreversible inhibitor of steroid sulfatase activity will allow the roles that these enzymes have in a number of physiological and pathological processes to be evaluated and also help to identify the active site of this enzyme.

Original languageEnglish
Pages (from-to)11508-11514
Number of pages7
JournalBiochemistry
Volume34
Issue number36
DOIs
Publication statusPublished - 12 Sep 1995

Cite this

PUROHIT, A ; Williams, G J ; HOWARTH, N M ; Potter, B V L ; REED, M J . / Inactivation of Steroid Sulfatase by an Active Site-Directed Inhibitor, Estrone-3-O-Sulfamate. In: Biochemistry. 1995 ; Vol. 34, No. 36. pp. 11508-11514.
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abstract = "Steroid sulfatases are responsible for the hydrolysis of 3 beta-hydroxy steroid sulfates, such as cholesterol and pregnenolone sulfate, and have an important role in regulating the synthesis of estrogenic steroids, from estrone sulfate and dehydroepiandrosterone sulfate, in endocrine-dependent tumors. Although little is known about the mechanism by which the sulfate group is removed from a steroid nucleus, an active site-directed sulfatase inhibitor has been developed. This inhibitor, estrone-3-O-sulfamate (EMATE), was synthesized by treating the sodium salt of estrone with sulfamoyl chloride. This compound inhibited not only estrone sulfatase but also dehydroepiandrosterone sulfatase activity in placental microsomes and in intact MCF-7 breast cancer cells. Pretreatment of MCF-7 cells or placental microsomes with EMATE, followed by extensive washing or dialysis indicated irreversible inhibition. This was confirmed by showing that EMATE inhibited estrone sulfatase activity in placental microsomes in a time-, concentration-, and pH-dependent manner. The enzyme is protected from inactivation by estrone sulfate, which is also consistent with active site-directed inhibition. EMATE is proposed to inactivate estrone sulfatase by irreversible sulfamoylation of the enzyme. Maximum enzyme activity was detected at pH 8.6, and the maximum rate of enzyme inactivation by EMATE also occurred at this pH. The pK(a) values of the enzymatic reaction and pK(a) of inactivation were 7.2 and 9.8, providing evidence that two active site residues are being modified by EMATE. As the phenolic pK(a) of tyrosine (9.7) and the pK(a) of histidine (6.8) are similar to the pK(a) values of inactivation, these amino acid residues may play a role in the catalytic mechanism. The development of an active site-directed irreversible inhibitor of steroid sulfatase activity will allow the roles that these enzymes have in a number of physiological and pathological processes to be evaluated and also help to identify the active site of this enzyme.",
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Inactivation of Steroid Sulfatase by an Active Site-Directed Inhibitor, Estrone-3-O-Sulfamate. / PUROHIT, A ; Williams, G J ; HOWARTH, N M ; Potter, B V L ; REED, M J .

In: Biochemistry, Vol. 34, No. 36, 12.09.1995, p. 11508-11514.

Research output: Contribution to journalArticle

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T1 - Inactivation of Steroid Sulfatase by an Active Site-Directed Inhibitor, Estrone-3-O-Sulfamate

AU - PUROHIT, A

AU - Williams, G J

AU - HOWARTH, N M

AU - Potter, B V L

AU - REED, M J

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AB - Steroid sulfatases are responsible for the hydrolysis of 3 beta-hydroxy steroid sulfates, such as cholesterol and pregnenolone sulfate, and have an important role in regulating the synthesis of estrogenic steroids, from estrone sulfate and dehydroepiandrosterone sulfate, in endocrine-dependent tumors. Although little is known about the mechanism by which the sulfate group is removed from a steroid nucleus, an active site-directed sulfatase inhibitor has been developed. This inhibitor, estrone-3-O-sulfamate (EMATE), was synthesized by treating the sodium salt of estrone with sulfamoyl chloride. This compound inhibited not only estrone sulfatase but also dehydroepiandrosterone sulfatase activity in placental microsomes and in intact MCF-7 breast cancer cells. Pretreatment of MCF-7 cells or placental microsomes with EMATE, followed by extensive washing or dialysis indicated irreversible inhibition. This was confirmed by showing that EMATE inhibited estrone sulfatase activity in placental microsomes in a time-, concentration-, and pH-dependent manner. The enzyme is protected from inactivation by estrone sulfate, which is also consistent with active site-directed inhibition. EMATE is proposed to inactivate estrone sulfatase by irreversible sulfamoylation of the enzyme. Maximum enzyme activity was detected at pH 8.6, and the maximum rate of enzyme inactivation by EMATE also occurred at this pH. The pK(a) values of the enzymatic reaction and pK(a) of inactivation were 7.2 and 9.8, providing evidence that two active site residues are being modified by EMATE. As the phenolic pK(a) of tyrosine (9.7) and the pK(a) of histidine (6.8) are similar to the pK(a) values of inactivation, these amino acid residues may play a role in the catalytic mechanism. The development of an active site-directed irreversible inhibitor of steroid sulfatase activity will allow the roles that these enzymes have in a number of physiological and pathological processes to be evaluated and also help to identify the active site of this enzyme.

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DO - 10.1021/bi00036a025

M3 - Article

VL - 34

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EP - 11514

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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