Affinity labeling of aryl sulfotransferase IV. Identification of a peptide sequence at the binding site for 3'-phosphoadenosine-5'-phosphosulfate

Yuqun Zheng; Alan Bergold; Michael W Duffel

doi:10.1016/S0021-9258(18)43814-8

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Affinity labeling of aryl sulfotransferase IV. Identification of a peptide sequence at the binding site for 3'-phosphoadenosine-5'-phosphosulfate

Journal article

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Affinity labeling of aryl sulfotransferase IV. Identification of a peptide sequence at the binding site for 3'-phosphoadenosine-5'-phosphosulfate

Yuqun Zheng, Alan Bergold and Michael W Duffel

The Journal of biological chemistry, Vol.269(48), pp.30313-30319

12/02/1994

DOI: 10.1016/S0021-9258(18)43814-8

PMID: 7982943

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Abstract

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.

Adenosine Triphosphate - analogs & derivatives

Adenosine Triphosphate - metabolism

Adenosine Triphosphate - pharmacology

Affinity Labels

Amino Acid Sequence

Animals

Arylsulfotransferase - chemistry

Arylsulfotransferase - isolation & purification

Arylsulfotransferase - metabolism

Binding Sites

Chromatography, High Pressure Liquid

DNA, Complementary - metabolism

Kinetics

Liver - enzymology

Molecular Sequence Data

Peptide Fragments - chemistry

Peptide Fragments - isolation & purification

Phosphoadenosine Phosphosulfate - metabolism

Rats

Recombinant Proteins - chemistry

Recombinant Proteins - isolation & purification

Recombinant Proteins - metabolism

Sequence Homology, Amino Acid

Sulfotransferases - chemistry

Details

Title: Subtitle: Affinity labeling of aryl sulfotransferase IV. Identification of a peptide sequence at the binding site for 3'-phosphoadenosine-5'-phosphosulfate
Creators: Yuqun Zheng - University of Iowa
Alan Bergold - University of Iowa
Michael W Duffel - University of Iowa
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.269(48), pp.30313-30319
DOI: 10.1016/S0021-9258(18)43814-8
PMID: 7982943
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Grant note: R01 CA038683 / NCI NIH HHS CA38683 / NCI NIH HHS
Language: English
Date published: 12/02/1994
Academic Unit: Pharmaceutical Sciences and Experimental Therapeutics; Medicinal and Natural Products Chemistry
Record Identifier: 9984303287202771

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