Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7-azido-4-methylcoumarin.
A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 +/- 0.05 mM). Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.
Chen
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G
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Battaglia
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E
Senay
C
C
Falany
C N
CN
Radominska-Pandya
A
A
D000595:Amino Acid Sequence; D000818:Animals; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003374:Coumarins / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D019920:Photoaffinity Labels / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
C097886:7-azido-4-methylcoumarin; D003374:Coumarins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D019920:Photoaffinity Labels; D015239:Arylsulfotransferase