A P-loop related motif (GxxGxxK) highly conserved in sulfotransferases is required for binding the activated sulfate donor.
A nucleotide binding motif termed the P-loop has been described for ATP- and GTP-binding proteins. The primary structure typically consists of a glycine-rich region followed by a conserved lysine. A related structure (GxxGxxK) noted in sulfotransferases has been suggested to be important for the binding of the cofactor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the universal sulfate donor for this class of enzymes. Using molecular techniques, point mutations that substituted alanines for the putative critical residues were introduced into the cDNA for estrogen sulfotransferase. The altered construct, although fully expressed by Chinese hamster ovary-K1 cells, demonstrated negligible enzymatic activity and failed to photoaffinity label with [35S]PAPS. In contrast, a construct in which three other amino acids in the region of the P-loop motif were similarly mutated retained activity and was photoaffinity labeled with [35S]PAPS. These data strongly support the notion that the P-loop motif found in all cloned sulfotransferases constitutes, at least in part, the PAPS binding site for these enzymes.
Komatsu
K
K
Section on Steroid Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Driscoll
W J
WJ
Koh
Y C
YC
Strott
C A
CA
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002417:Cattle; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006224:Cricetinae; D006168:Guinea Pigs; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014162:Transfection
D000345:Affinity Labels; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; C020122:estrone sulfotransferase