Journal article
Studies on an affinity label for the sulfuryl acceptor binding site in an aryl sulfotransferase
Chemico-biological interactions, Vol.109(1), pp.81-92
1998
DOI: 10.1016/S0009-2797(97)00122-1
PMID: 9566735
Abstract
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.
Details
- Title: Subtitle
- Studies on an affinity label for the sulfuryl acceptor binding site in an aryl sulfotransferase
- Creators
- Michael W Duffel - University of IowaGuangping Chen - University of IowaVyas Sharma - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Chemico-biological interactions, Vol.109(1), pp.81-92
- Publisher
- Elsevier Ireland Ltd
- DOI
- 10.1016/S0009-2797(97)00122-1
- PMID
- 9566735
- ISSN
- 0009-2797
- eISSN
- 1872-7786
- Language
- English
- Date published
- 1998
- Academic Unit
- Pharmaceutical Sciences and Experimental Therapeutics; Medicinal and Natural Products Chemistry
- Record Identifier
- 9984303286702771
Metrics
7 Record Views