PubMed2000
Human monoamine (M) form and simple phenol (P) form phenol sulfotransferases (PSTs) are greater than 93% identical in their primary sequences and yet display distinct substrate specificities and other enzymatic properties. Through the generation and characterization of a series of chimeric PSTs, we have previously demonstrated two highly variable regions within their sequences to be responsible for determining their substrate phenotypes (Sakakibara, Y., Takami, Y., Nakayama, T., Suiko, M., and Liu, M.-C. (1998) J. Biol. Chem. 273, 6242-6247). By employing the site-directed mutagenesis technique, the present study aims to identify and quantitatively evaluate the specific amino acid residues critical to the substrate binding and catalysis in these two enzymes. Twelve mutated M-PSTs and seven mutated P-PSTs were generated, expressed, and purified. Enzymatic characterization showed that, of the twelve mutated M-PSTs, mutations at residues Asp-86, Glu-89, and Glu-146 resulted in a dramatic decrease in V(max)/K(m) with dopamine as substrate, being greater than 450 times for the D86A/E89I/E146A mutated M-PST. With p-nitrophenol as substrate, the V(max)/K(m) determined for the D86A/E89I/E146A-mutated M-PST increased more than 25 times and approached that determined for the wild-type P-PST. These results indicated that the concerted action of the three mutated residues (D86A, E89I, and E146A) is sufficient for the conversion of the substrate phenotype of M-PST to that of P-PST. Among the mutated P-PSTs, the I89E- and A146E-mutated P-PSTs displayed considerable deviations in V(max)/K(m) with dopamine or p-nitrophenol as substrate. No corresponding changes, however, were detected with the opposite compound as substrate. These results indicated that, in contrast to M-PST, mutations at Ala-86, Ile-89, and Ala-146 to the corresponding residues in M-PST are not sufficient for rendering the change of P-PST substrate phenotype to that of M-PST. For both M-PSTs and P-PSTs, mutations at Lys-48 or His-108 led to the loss of sulfotransferase activities, indicating their importance in the catalytic mechanism.