Yq. Zheng et al., AFFINITY LABELING OF ARYL SULFOTRANSFERASE-IV - IDENTIFICATION OF A PEPTIDE SEQUENCE AT THE BINDING-SITE FOR 3'-PHOSPHOADENOSINE-5'-PHOSPHOSULFATE, The Journal of biological chemistry, 269(48), 1994, pp. 30313-30319
(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.