Arginine residues in the active site of human phenol sulfotransferase (SULT1A1)

Abstract

Cytosolic sulfotransferases (STs) catalyze the sulfation of hydroxyl containing compounds. Human phenol sulfotransferase (SULT1A1) is the major human ST that catalyzes the sulfation of simple phenols. Because of its broad substrate specificity and lack of endogenous substrates, the biological function of SULT1A1 is believed to be an important detoxification enzyme. In this report, amino acid modification, computer structure modeling, and site-directed mutagenesis were used for studies of Arg residues in the active site of SULT1A1. The Arg-specific modification reagent, 2,3-butanedione, inactivated SULT1A1 in an efficient, time- and concentration-dependent manner, suggesting Arg residues play an important role in the catalytic activity of SULT1A1. According to the computer model, Arg78, Arg130, and Arg257 may be important for SULT1A1 catalytic activity. Site-directed mutagenesis results demonstrated that the positive charge on Arg78 is not critical for SULT1A1 because R78A is still active. In contrast, a negative charge at this position, R78E, completely inactivated SULT1A1. Arg78 is in close proximity to the site of sulfuryl group transfer. Arg257 is located very close to the 3'-phosphate in adenosine 3'-phosphate 5'-phosphosulfate (PAPS). Site-directed mutagenesis demonstrated that Arg257 is critical for SULT1A1: both R257A and R257E are inactive. Although Arg130 is also located very close to the 3'-phosphate of PAPS, R130A and R130E are still active, suggesting that Arg130 is not a critical residue for the catalytic activity of SULT1A1. Computer modeling suggests that the ionic interaction between the positive charge on Arg257, and the negative charge on 3'-phosphate is the primary force stabilizing the specific binding of PAPS.

Fig. 1

Chemical reaction for 2,3-butanedione inactivation of Arg residues in a protein.

Fig. 2. Time- and concentration-dependent inactivation of SULT1A1 by 2,3-butanedione

A, log(percent activity) versus time. SULT1A1 (0.1 mg/ml) was incubated with different concentrations of 2,3-butanedione at room temperature in 50 mM borate, 1 M NaCl, pH 7.5. Aliquots were taken at different time points. SULT1A1 remaining activity was assayed using the PNPS assay method in a mixture containing 0.5 M arginine. B, log(K_app) versus log(BD).

Fig. 3. Kinetic parameter determination of partially inactivated SULT1A1

The PNPS assay method was used for the determination of enzyme activity. A, varied substrate 4-phenylphenol concentration. B, varied PAPS concentration.

Fig. 4. PAP and substrate protection of 2,3-butanedione inactivation of SULT1A1

The PNPS assay method was used to determine the remaining enzyme activity.

Fig. 5. Computer modeling structure of SULT1A1

The structure was depicted by RasMol 2.6. The whole structure was displaced by “Backbone.” PAPS, 2-naphthol, Arg⁷⁸, Arg¹³⁰, Arg²⁵⁷, and Arg²¹³ were displaced by “Spacefill.”

Fig. 6. SDS-PAGE of the purified SULT1A1 mutant proteins

12% acrylamide gel was used for the separation of proteins. The gel was run at 200 V.

Fig. 7. 2-Naphthol sulfation activity of SULT1A1 mutant proteins

The methylene blue assay method was used for enzyme activity determinations. 0.1 M 2-naphthol was used as substrate for the assay at 8 μM PAPS, pH 6.2.

Fig. 8. Computer modeling structure of SULT1A1 showing Arg⁷⁸ and related residues and ligands

The model structure of SULT1A1 was depicted by RasMol 2.6. Only Arg⁷⁸, PAPS, 2-naphthol, Lys¹⁰⁶, and Asp⁵⁹ are displaced.

Fig. 9. Computer modeling structure of SULT1A1 showing charged residues in close proximity to 3′-phosphate of PAPS

The model structure of SULT1A1 was depicted by RasMol 2.6. Only PAPS and charged residues within 5 Å to 3′-phosphate are displaced.