Studies on an affinity label for the sulfuryl acceptor binding site in an aryl sulfotransferase.
Active site-directed affinity labeling was utilized to elucidate peptide sequences at the binding site for sulfuryl acceptors in rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9). The affinity labeling reagent, N-bromoacetyl-4-hydroxyphenylamine, was designed on the basis of substrate specificity studies with para-substituted phenols, utilization of a bromoacetamido group for reactivity with active site amino acid residues and its similarity to acetaminophen, a known substrate for aryl (phenol) sulfotransferases. AST IV utilized N-bromoacetyl-4-hydroxyphenylamine as a substrate with kinetic constants that compared favorably to those obtained with acetaminophen. Incubation of AST IV with N-bromoacetyl-4-hydroxyphenylamine at pH 7.0 in the absence of PAPS and other substrates resulted in an irreversible inactivation of the enzyme that was both time- and concentration-dependent. [14C]-N-bromoacetyl-4-hydroxyphenylamine was synthesized and used to analyze the regions of protein sequence that were involved in the binding of the affinity label. AST IV was incubated with [14C]-N-bromoacetyl-4-hydroxyphenylamine, hydrolyzed with endoproteinase Lys-C and the labeled peptides were purified by HPLC. Control incubations of AST IV with the affinity label in the presence of 4-propylphenol and PAP were utilized to ascertain the specificity of the interaction. Sequence analysis of the labeled peptides, carried out by automated Edman degradation, revealed labeling sites on cysteine (Cys-232, Cys-283 and Cys-289) and lysine (Lys-286) residues near the C-terminus of the protein. The locations of these labeling sites were further evaluated both by sequence-alignment with other sulfotransferases and by theoretical calculations on predicted secondary structure.
Duffel
M W
MW
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of Iowa, Iowa City 52242, USA. michael-duffel@uiowa.edu
Chen
G
G
Sharma
V
V
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D051381:Rats; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000378:metabolism*; D013457:Sulfur Compounds / Q000378:metabolism
D000345:Affinity Labels; D013457:Sulfur Compounds; D015238:Sulfotransferases; C077557:tyrosine-ester sulfotransferase