Affinity labeling of aryl sulfotransferase IV. Identification of a peptide sequence at the binding site for 3'-phosphoadenosine-5'-phosphosulfate.
2'-O-[(R)-Formyl(adenin-9-yl)-methyl]-(S)-glyceraldehyde 3'-triphosphate (also designated as ATP dialdehyde or ATPDA) was utilized as an affinity label for the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding site of an aryl sulfotransferase. The sulfotransferase employed in these studies was rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9), for which a cDNA had been previously cloned and expressed in Escherichia coli and the resulting enzyme purified to homogeneity. ATPDA was a time-dependent irreversible inhibitor of the recombinant AST IV, and this inhibition was prevented by including either PAPS or adenosine 3',5'-diphosphate (PAP) in the incubation of AST IV with ATPDA. Experiments relating covalent binding of [2,8-3H]ATPDA with catalytic activity indicated that 1 nmol of the affinity label was bound per nmol of AST IV subunit. Incubation of [2,8-3H]ATPDA with the enzyme followed by reduction with sodium cyanoborohydride, proteolysis with trypsin, and separation of the resulting peptides by high pressure liquid chromatography yielded two labeled peptide fractions. Automated sequence analysis showed that both modified peptide fractions were derived from the same sequence in AST IV: 63-Leu-Glu-Lys-Cys-Gly-Arg-68. Both the sequencing results and examination of the two peptide fractions by matrix-assisted laser desorption ionization mass spectrometry indicated that the ATPDA affinity label was bound to the hexapeptide at both lysine 65 and cysteine 66. These affinity labeled amino acids are located within a region of sequence in AST IV that shows considerable homology with various sulfotransferases that possess diverse specificities for acceptor substrates, and this may provide insight into PAPS binding in other sulfotransferases.
Zheng
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Y
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242.
Bergold
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A
Duffel
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MW
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D015239:Arylsulfotransferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015238:Sulfotransferases / Q000737:chemistry
D000345:Affinity Labels; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010724:Phosphoadenosine Phosphosulfate; C017199:2',3'-dialdehyde ATP; D000255:Adenosine Triphosphate; D015238:Sulfotransferases; D015239:Arylsulfotransferase