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
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.