Structural characterization of human aryl sulphotransferases

Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greate r than 90% sequence identity, but vary markedly in their ability to catalys e the sulphonation of dopamine and p-nitrophenol. In order to investigate t he amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes t wo regions of high sequence divergence, termed Regions A (amino acids 44-10 7) and B (amino acids 132-164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to chang e 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specific ity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single am ino acid change. However, compared with wild-type HAST1, a number of the mu tations resulted in interference with substrate binding, as shown by elevat ed K-i values towards the co-substrate 3'-phosphoadenosine 5'-phosphosulpha te. and in some cases loss of activity towards dopamine. These findings sug gest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopam ine, whereas a single amino acid at position 146 determines p-nitrophenol a ffinity. A HAST 1 mutant was constructed to express a protein with four ami no acids deleted (P87-P90). These amino acids were hypothesized to correspo nd to a loop region in close proximity to the substrate-binding pocket. Int erestingly, the protein showed substrate specificities more similar to wild -type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.