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Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7-Azido-4-methylcoumarin

Published online by Cambridge University Press:  01 October 1999

GUANGPING CHEN ,
ERIC BATTAGLIA ,
CLAIRE SENAY ,
CHARLES N. FALANY  and
ANNA RADOMINSKA-PANDYA
GUANGPING CHEN
Affiliation:
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
ERIC BATTAGLIA
Affiliation:
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Present address: Gastroenterology Division, Brigham and Women's Hospital, Boston, MA 02115.
CLAIRE SENAY
Affiliation:
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Present address: UMR 7561, CNRS-Faculty of Medicine, University Henri Poincaré Nancy 1, 54000 Vandoeuvre-les-Nancy, France.
CHARLES N. FALANY
Affiliation:
Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294
ANNA RADOMINSKA-PANDYA
Affiliation:
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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Abstract

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.

Keywords

fluorescent probephenol sulfotransferasesphotoaffinity labelingstructure–functionsubstrate binding site
Type
Research Article
Information
Protein Science , Volume 8 , Issue 10 , October 1999 , pp. 2151 - 2157
Copyright
© 1999 The Protein Society

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