Enzymatic sulfation of biomolecules

Details Enzymatic sulfation of biomolecules Enzymatic sulfation of biomolecules

1999-09-10

2001-07-03

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing sulfur-containing organic compound

C435S101000, C435S193000, C435S257300, C435S320100, C536S023200

Reexamination Certificate

active

06255088

ABSTRACT:

TECHNICAL FIELD
The present invention relates to the preparative enzymatic synthesis of sulfated biomolecules. More particularly, the present invention relates to the preparative enzymatic synthesis of sulfated biomolecules including carbohydrates, oligosaccharides, peptides, proteins, flavonoids, and steroids and to xenobiotic sulfations. The present invention also relates to continuous assays for the spectrophotometric analysis of sulfotransferase reactions.
BACKGROUND
Sulfotransferases comprise a family of enzymes that catalyze the transfer of a sulfonate or sulfuryl group (SO
3
31
) from the cofactor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) (1) to an acceptor molecule. Even though it is more accurate to call these sulfonation reactions, the term sulfation is still widely used. The term sulfation will be employed within this specification with the understanding that a sulfonate or sulfuryl group is transferred. Sulfotransferases mediate sulfation of different classes of substrates such as carbohydrates, oligosaccharides, peptides, proteins, flavonoids, and steroids for a variety of biological functions including signaling and modulation of receptor binding (Bowman, K. G., et al., (1999)
Chem. Biol.
6, R9-R22; and Falany, C. N., (1997)
FASEB J.
11, 1-2). Within the past three years, many new sulfotransferases have been identified and cloned (Aikawa, J.-I., et al., (1999)
J. Biol. Chem.
274, 2690; Dooley, T. P., (1998)
Chemico-Biol. Interact.
109, 29; Fukuta, M., et al. (1998)
Biochim. Biophys. Act.
1399, 57; Habuchi, H., et al., (1998)
J. Biol. Chem.
273, 9208; Mazany, K. D., et al., (1998)
Biochim. Biophys. Act.
1407, 92; Nastuk, M. A., et al. (1998)
J. Neuroscience
18, 7167; Ong, E., et al., (1998)
J. Biol. Chem.
273, 5190; Ouyang, Y.-B., et al., (1998)
J. Biol. Chem.
273, 24770; Saeki, Y., et al. (1998)
J. Biochem.
124, 55; Uchimura, K., et al. (1998)
J. Biol. Chem.
273, 22577; and Yoshinari, K., et al., (1998)
J. Biochem.
123, 740). A facile means to produce large amounts of sulfated product and efficient sulfotransferase assays are essential for the biological study of these enzymes and their sulfated products.
Sulfotransferases are the family of enzymes catalyzing sulfotransfer reactions, or the transfer of a sulfuryl group (SO
3
) from 3′-phosphoadenosine-5′-phosophosulfate (PAPS) to an acceptor molecule. Sulfotransferases, present in most organisms and in all human tissues, mediate sulfation of different classes of acceptors for a variety of biological functions. To date, more than 30 sulfotransferase cDNAs have been isolated from animal, plant, and bacterial sources (Weinshilboum, R. M., et al. (1997)
FASEB J.
11, 3-14). The varied and important roles sulfotransferases play in biological systems have only recently been uncovered, including detoxification, cell signaling, and modulation of receptor binding (Bowman, K. G., et al. (1999)
Chern. Biol.
6, R9-R22; and Falany, C. N., (1997)
FASEB J.
11, 206-216). Drug design to selectively inhibit these therapeutically important enzymes has quickly followed the discovery of their biological roles (Seah, V. M. Y., et al. (1994)
Biochem. Pharmacol.
47, 1743-9; Bartzatt, R., et al. (1994)
Biochem. Pharmacol.
47, 2087-95; Matsui, M., et al. (1995)
Biochem. Pharmacol.
49, 739-41; Wong, C.-K., et al. (1997)
Biochem. Biophys. Res. Commun.
233, 579-583; and Schuur, A. G., et al. (1998)
Chem. Res. Toxicol.
11, 1075-1081). Given the monumental speed with which new sulfotransferases are being identified and interest in their synthetic application and the development of sulfotransferase inhibitors, simple and rapid activity assays are vital to the progress of this field.
PAPS, the universal sulfate donor and source of sulfate for all sulfotransferases, is a highly expensive and unstable molecule that has been an obstacle to the large-scale production of enzymatically sulfated products. The half-life of PAPS in aqueous solution at pH 8.0 is approximately 20 hours and is available from Sigma Co. Product inhibition by adenosine 3′,5′-diphosphate (PAP) (3) has also been a limiting factor to large-scale applications. PAP inhibition of hydroxysteroid sulfotransferase was determined to be K
i
=14 &mgr;M ( Marcus, D. J., et al. (1980)
Aial. Biochem.
107, 296). PAP inhibits NodST with K
i
=0.1 &mgr;M (Lin, C.-H., et al., (1995)
J. Am. Cheni. Soc.
117, 8031).
SUMMARY
One aspect of the invention relates to an improved method for the preparative enzymatic sulfation of biomolecules, including carbohydrates, oligosaccharides, peptides, proteins, flavonoids, and steroids, and to xenobiotic sulfations, wherein the improvement is directed to the enzymatic regeneration of 3′-phosphoadenosine-5′- phosphosulfate (PAPS) from its byproduct 3′,5′-diphosphate (PAP) using a recombinant aryl sulfotransferase and p-nitrophenyl sulfate. This regeneration system is more convenient than the existing methods for the kinetic analysis and synthetic application of sulfotransferases.
Accordingly, one aspect of the invention is directed to an improved sulfation process. The process is of a type wherein the sulfation of a biomolecule is catalyzed by a sulfotransferase with a conversion of 3′-phosphoadenosine-5′-phosphosulfate to adenosine 3′,5′-diphosphate. The improvement is directed to the coupling of the sulfation process with an enzymatic regeneration of the 3′-phosphoadenosine-5′-phosphosulfate from the adenosine 3′,5′-diphosphate. The enzymatic regeneration employs an arylsulfotransferase as the catalyst and an aryl sulfate as a substrate. Preferred biomolecules include carbohydrates, oligosaccharides, peptides, proteins, flavonoids, and steroids. The invention may also be employed to enhance xenobiotic sulfation reactions employing PAPS dependent sulfotransferases.
Another aspect of the invention is directed to a continuous spectrophotometric coupled-enzyme assay is employed for assaying sulfotransferase activity. This assay is based on the regeneration of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) from the desulfated 3′-phosphoadenosine-5′-phosphate (PAP) by a recombinant aryl sulfotransferase using p-nitrophenyl sulfate as the sulfate donor and visible spectrophotometric indicator of enzyme turnover. Here recombinant rat aryl sulfotransferase IV (AST-IV) is expressed, resolved to the pure &bgr;-form during purification, and utilized for the regeneration. The activity of &bgr;AST-IV to catalyze the synthesis of PAPS from PAP and p-nitrophenyl sulfate is demonstrated via capillary zone electrophoresis, and the kinetics of this reverse-physiological reaction are calculated. &bgr;AST-IV is then applied to the coupled enzyme system, where the steady-state activity of the commercially available Nod factor sulfotranferase is verified with an enzyme concentration study and substrate-specificity assays of N-chitoses. The potential applications of this assay include rapid kinetic determinations for carbohydrate and protein sulfotransferases, high-throughput screening of potential sulfotransferase substrates and inhibitors, and biomedical screening of blood samples and other tissues for specific sulfotransferase enzyme activity and substrate concentration.
Accordingly, another aspect of the invention is directed to an assay for monitoring an enzymatic sulfation reaction. The enzymatic sulfation reaction is of the type which employs a conversion of 3′-phosphoadenosine-5′-phosphosulfate to adenosine 3′,5′-diphosphate. The assay includes two steps. In the first step, the enzymatic sulfation reaction is coupled with an enzymatic regeneration of the adenosine 3′,5′-diphosphate using an arylsulfotransferase in the presence of an aryl sulfate. Then, in the second step, the desulfation of the aryl sulfate is monotored spectroscopically and correlated with the enzymatic sulfation reaction of the first step.
Another aspect of the invention is directed

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