AFFINITY LABELING OF ARYL SULFOTRANSFERASE-IV - IDENTIFICATION OF A PEPTIDE SEQUENCE AT THE BINDING-SITE FOR 3'-PHOSPHOADENOSINE-5'-PHOSPHOSULFATE

(R)-Formyl(adenin-9-yl)-methyl]-(S)-glyceraldehyde 3'-triphosphate (al so designated as ATP dialdehyde or ATPDA) was utilized as an affinity label for the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding sit e 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 bee n previously cloned and expressed in Escherichia coli and the resultin g enzyme purified to homogeneity. ATPDA was a time-dependent irreversi ble inhibitor of the recombinant AST IV and this inhibition was preven ted by including either PAPS or adenosine 3',5'-diphosphate (PAP) in t he incubation of AST IV with ATPDA. Experiments relating covalent bind ing of [2,8-H-3]ATPDA with catalytic activity indicated that 1 nmol of the affinity label was bound per nmol of AST IV subunit. Incubation o f [2,8-H-3]ATPDA with the enzyme followed by reduction with sodium cya noborohydride, proteolysis with trypsin, and separation of the resulti ng peptides by high pressure liquid chromatography yielded two labeled peptide fractions. Automated sequence analysis showed that both modif ied peptide fractions were derived from the same sequence in AST IV: 6 3-Leu-Glu-Lys-Cys-Gly-Arg-68. Both the sequencing results and examinat ion of the two peptide fractions by matrix-assisted laser desorption i onization mass spectrometry indicated that the ATPDA affinity label wa s bound to the hexapeptide at both lysine 65 and cysteine 66. These af finity 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.