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 b een characterized for use in photoaffinity labeling of the substrate bindin g site of human phenol sulfotransferase (SULT1Al or P-PST-1). For the photo affinity labeling experiments, SULT1Al cDNA was expressed in Escherichia co li as a fusion protein to maltose binding protein (MBP) and purified to app arent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1Al and SULT1Al were efficiently photolabel ed with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1 Al (K-i = 0.47 +/- 0.05 mM). Moreover, enzyme activity toward 2-naphthol wa s inactivated in a time-and concentration-dependent manner. SULT1Al inactiv ation by AzMC was protected by substrate but was not protected by cosubstra te. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1Al. F urther studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.