Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: Site-directed mutagenesis and kinetic studies

Citation
La. Brix et al., Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: Site-directed mutagenesis and kinetic studies, BIOCHEM, 38(32), 1999, pp. 10474-10479
Citations number
17
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
38
Issue
32
Year of publication
1999
Pages
10474 - 10479
Database
ISI
SICI code
0006-2960(19990810)38:32<10474:AOTSSO>2.0.ZU;2-9
Abstract
Sulfonation is an important metabolic process involved in the excretion and in some cases activation of various endogenous compounds and xenobiotics. This reaction is catalyzed by a family of enzymes named sulfotransferases. The cytosolic human sulfotransferases SULT1A1 and SULT1A3 have overlapping yet distinct substrate specificities. SULT1A1 favors simple phenolic substr ates such as p-nitrophenol, whereas SULT1A3 prefers monoamine substrates su ch as dopamine. In this study we have used a variety of phenolic substrates to functionally characterize the role of the amino acid at position 146 in SULT1A1 and SULT1A3. First, the mutation A146E in SULT1A1 yielded a SULT1A 3-like protein with respect to the Michaelis constant for simple phenols. T he mutation E146A in SULT1A3 resulted in a SULT1A1-like protein with respec t to the Michaelis constant for both simple phenols and monoamine compounds . When comparing the specificity of SULT1A3 toward tyramine with that for p -ethylphenol (which differs from tyramine in having no amine group on the c arbon side chain), we saw a 200-fold preference for tyramine. The kinetic d ata obtained with the E146A mutant of SULT1A3 for these two substrates clea rly showed that this protein preferred substrates without an amine group at tached. Second, changing the glutamic acid at position 146 of SULT1A3 to a glutamine, thereby neutralizing the negative charge at this position, resul ted in a 360-fold decrease in the specificity constant for dopamine. The re sults provide strong evidence that residue 146 is crucial in determining th e substrate specificity of both SULT1A1 and SULT1A3 and suggest that there is a direct interaction between glutamic acid 146 in SULT1A3 and monoamine substrates.