From the Biomedical Research Center, The University of Texas Health Center, Tyler, Texas 75708
Received for publication, April 1, 2002, and in revised form, November 5, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human monoamine-form phenol
sulfotransferase (PST), SULT1A3, has a unique
3,4-dihydroxyphenylalanine (Dopa)/tyrosine-sulfating activity
that is stereospecific for their D-form enantiomers
and can be stimulated dramatically by Mn2+. This activity
is not present in the simple phenol-form PST, SULT1A1, which is
otherwise >93% identical to SULT1A3 in amino acid sequence. The
majority of the differences between these two proteins reside in two
variable regions of their sequences. Through the characterization of
chimeric PSTs where these two regions were exchanged between them, it
was demonstrated that variable Region II of SULT1A3 is required for the
stereospecificity of its Dopa/tyrosine-sulfating activity, whereas
variable Region I of SULT1A3 is required for the stimulation by
Mn2+ of this activity. Further studies using point-mutated
SULT1A3s mutated at amino acid residues in these two regions and
deletional mutants missing residues 84-86 and 84-90 implicate residue
Glu-146 (in variable Region II of SULT1A3), as well as the
presence of residues 84-90 of variable Region I, in the
stereospecificity in the absence of Mn2+. Residue Asp-86
(in variable Region I of SULT1A3), on the other hand, is critical in
the Mn2+ stimulation of the Dopa/tyrosine-sulfating
activity of SULT1A3. A model is proposed, with reference to the
reported x-ray crystal structure of SULT1A3, to explain how the normal
role of SULT1A3 in dopamine regulation may be subverted in the presence
of Mn2+. These studies could be relevant in understanding
the stereoselective action of SULT1A3 on chiral drugs.
The sulfotransferases
(STs),1 which are ubiquitous
in both plants and animals, catalyze the sulfation of hydroxyl or amino groups on a variety of target acceptor molecules (1, 2). These enzymes
all use adenosine 3'-phosphate,5'-phosphosulfate (PAPS) as the sulfonyl
group donor (3) and share sequences responsible for PAPS binding (4).
Although the membrane-bound STs use proteins, glycolipids, and other
macromolecules as acceptor substrates, the cytosolic STs sulfate
smaller molecules and are part of the Phase II detoxification pathway
for the biotransformation/excretion of drugs and xenobiotics (1, 2).
Increasingly, the cytosolic STs have also been shown to be important in
regulating the levels and/or activities of endogenous compounds such as
thyroid and steroid hormones, catecholamines, and bile acids (5,
6).
Based on their sequences, the cytosolic STs have been classified into
several gene families (4). Two human cytosolic STs, the monoamine-form
and the simple phenol-form phenol sulfotransferases, named
SULT1A3 and SULT1A1, respectively (4), show an extensive (>93%)
identity in their amino acid sequences (cf. Fig.
1A) and yet vary widely in their substrate specificity and
other properties (7-9). They have thus served as an ideal model system
to study structure/function relationships in these proteins.
Examination of their aligned sequences revealed that most of the
differences between SULT1A3 and SULT1A1 occur in two variable regions,
designated Region I (encompassing amino acid residues 84-89) and
Region II (including residues 143-148) (cf. Fig.
1A). Based on the hypothesis that the differences in these
two variable regions may account for the distinct properties of SULT1A3
and SULT1A1, we had prepared chimeric proteins (7), where these two
regions were reciprocally exchanged (cf. Fig.
1B). Characterization of these chimeras indicated that both
Regions I and II were indeed critical for the specificity of SULT1A3
for dopamine and of SULT1A1 for p-nitrophenol (7). To extend
the study further, we and others (8, 10, 11) had, by site-directed
mutagenesis, exchanged amino acid residues in these two regions between
SULT1A3 and SULT1A1. Results from these studies implicated residue 146 in Region II and residues 86 and 89 in Region I, as important in
determining the specificity of the enzymes for their respective substrates.
SULT1A3 is present in brain where it is believed to sulfate monoamine
neurotransmitters (particularly dopamine) with high activity and thus
to regulate their levels (5). It also serves a detoxifying function in
the intestine, where it may detoxify potentially lethal dietary
monoamines (12). Besides the activity toward its physiological
substrate, dopamine, for which it has a Km of 2 µM, SULT1A3 has been shown recently to display a unique
Dopa/tyrosine-sulfating activity at higher (millimolar and
sub-millimolar) concentrations of these substrates, which can be
dramatically stimulated by Mn2+ (13).
In this study, chimeras and site-directed mutants were used to explore
the structural basis for the stereospecificity and Mn2+
stimulation of the Dopa/tyrosine-sulfating activity of SULT1A3. SULT1A3
is present as a homodimer in its native state (14). Kinetic studies of
the Dopa/tyrosine-sulfating activity of SULT1A3 (15) suggested that, in
the case of D-tyrosine, where a 4-hydroxyphenyl group is to
be sulfated, the tyrosine-Mn2+ and
tyrosine-Mn2+-tyrosine complexes may be the real and
obligatory substrates. In the case of D-Dopa, where we had
shown that sulfation takes place exclusively at the 3-hydroxyphenyl
group (16), the substrate-Mn2+ complexes appeared to be
better substrates than D-Dopa. These kinetic data implied
that Mn2+, while interacting with the carboxyl group of the
Dopa/tyrosine substrate to form the complex, also coordinates with
amino acid residues on the enzyme. Previous x-ray crystallography study
on SULT1A3 (17) has suggested that residues 84-92 corresponding to the
variable Region I (cf. Fig. 1A) form a
"mobile" loop. Based on the amino acid residue in SULT1A3
identified as interacting with Mn2+ in the present study, a
model has been proposed, in reference to the above-mentioned mobile
loop, to explain how Mn2+ exerts its stimulatory effect on
the sulfation of tyrosine and Dopa by the enzyme. This study also
identified the amino acid residues and regions responsible for the
unusual stereospecificity of SULT1A3 for the D-enantiomers
of Dopa and tyrosine. The model developed may prove useful in
understanding the stereoselective action of SULT1A3 on chiral drugs
(18-21) and the possible effects of metal ions on this activity.
Materials--
L-Tyrosine, D-tyrosine,
L-Dopa, D-Dopa, ATP,
3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic
acid (TAPS), SDS, dithiothreitol, EDTA (tetrasodium salt),
isopropyl Preparation of Purified Wild-type and Chimeric
SULT1A3/SULT1A1 and Point-mutated SULT1A3s--
Wild-type
and chimeric SULT1A3 and SULT1A1, cloned/generated and expressed using
the pGEX-2TK glutathione S-transferase gene fusion system,
were purified using glutathione-Sepharose in conjunction with thrombin
cleavage to separate the fusion protein, based on the procedure
established previously (7). Point-mutated SULT1A3s were prepared using
the QuikChange site-directed mutagenesis kit, expressed using the
pGEX-2TK glutathione S-transferase gene fusion system and purified with
glutathione-Sepharose followed by thrombin cleavage to separate the
fusion protein, as described previously (8).
Generation, Expression, and Purification of Deletional Mutants of
SULT1A3--
The QuikChange site-directed mutagenesis kit from
Stratagene was used for the generation of deletional mutants of
SULT1A3. Briefly, wild-type SULT1A3 cDNA packaged in pGEX-2TK
prokaryotic expression vector was used as the template in conjunction
with specific mutagenic primers. To prepare the deletional SULT1A3 mutant lacking residues 84, 85, and 86 of the mobile loop (see Introduction), the mutagenic oligonucleotide primer set,
5'-CGGGTGCCCTTCCTTGAGCCAGGGGAACCCTCAGGG-3' and
5'-CCCTGAGGGTTCCCCTGGCTCAAGGAAGGGCACCCG-3', was used. Similarly, a
deletional mutant of SULT1A3 lacking residues 84 through 90 of the loop
was prepared using the mutagenic primer set,
5'-CGGGTGCCCTTCCTTGAGTCAGGGCTGGAGACTCTG-3' and
5'-CAGAGTCTCCAGCCCTGACTCAAGGAAGGGCACCCG-3'. The amplification conditions were 12 cycles of 30 s at 95 °C, 1 min at 55 °C,
and 15 min at 68 °C. The deletional SULT1A3 mutant sequences were verified by nucleotide sequencing (23). pGEX-2TK vector harboring individual deletional SULT1A3 mutant sequence was transformed into
competent XL1-Blue E. coli cells. The transformed cells, grown to A600 nm = ~0.5 in 1 liter of LB
medium supplemented with 100 µg/ml ampicillin and induced with 0.1 mM isopropyl Enzymatic Assay--
Sulfotransferase activities of the purified
wild-type and chimeric SULT1A3/SULT1A1, as well as point and deletional
mutant SULT1A3s, were assayed using [35S]PAPS as the
sulfonate group donor. The standard assay mixture, in a final volume of
25 µl, contained 50 mM TAPS buffer, pH 8.25, 15 µM [35S]PAPS, 5 mM
D- or L-tyrosine, or 1 mM
D- or L-Dopa without additions (control)
or with 1 mM EDTA, 5 mM MnCl2, or 5 mM EDTA plus 5 mM MnCl2. The enzyme
dilutions were prepared in 50 mM TAPS, pH 8.25, containing
10% glycerol and 8 mM dithiothreitol. The reaction was
started by the addition of 5 µl of the enzyme preparation, allowed to
proceed for 3 or 10 min (so that the reaction reached no more than
5-10% of completion) at 37 °C, and terminated by heating at
100 °C for 2 min. The precipitates were cleared by centrifugation
for 1 min, and the supernatant was subjected to the analysis of
[35S]sulfated D- or L-tyrosine or
D- or L-Dopa based on the TLC procedure established previously (24). In the experiments with D- or
L-tyrosine as substrate, the sulfated product was separated
by ascending TLC on a cellulose TLC plate in a solvent system
containing n-butanol, isopropanol, 88% formic acid, water
in a 3:1:1:1 ratio by volume. In the experiments with D- or
L-Dopa as substrate, the sulfated product was first
subjected to high voltage (1000 volts) thin-layer electrophoresis in
one dimension before performing the ascending TLC separation in the
second dimension. Each experiment was performed in triplicate, together
with a control without enzyme. The results obtained were calculated and
expressed in nmol sulfated product formed/min/mg protein.
Miscellaneous Methods--
[35S]PAPS was
synthesized from ATP and carrier-free [35S]sulfate using
the bifunctional human ATP sulfurylase/ATP sulfurylase/adenosine 5'-phosphosulfate kinase, and its purity was determined as described previously (25). The [35S]PAPS synthesized was then
adjusted to the required concentration and specific activity by the
addition of cold PAPS. SDS-polyacrylamide gel electrophoresis was
performed on 12% polyacrylamide gels using the method of Laemmli (26).
Protein determination was based on the method of Bradford with bovine
serum albumin as standard (27).
Despite recent efforts from several laboratories (7-11), there is
still relatively scant information concerning the structure/function relationships of cytosolic ST enzymes. As noted earlier, SULT1A3 and
SULT1A1, which are >93% identical in amino acid sequence and yet
display distinct substrate specificity and other properties, provide an
excellent model for studies in this regard. In contrast to SULT1A1,
SULT1A3 exhibits two unique activities. The sulfating activity toward
dopamine, which presumably helps to regulate the levels of this
endogenous compound, has a pH optimum of ~7.0 and a
Km of 2 µM (in the physiological
range) (14, 28). Mn2+ stimulated this activity to a
comparatively smaller extent (~2- to 3-fold) (13) but appeared to
increase the Km for dopamine slightly (15). The
Dopa/tyrosine-sulfating activity of SULT1A3, on the other hand, has the
hallmarks of a detoxifying activity, with a pH optimum between 8 and 9 (29) and a Km (if we consider the substrate rather
than substrate-Mn2+ complex) in the millimolar or
sub-millimolar range (7, 13). In contrast to the dopamine-sulfating
activity, the Dopa/tyrosine-sulfating activity can be stimulated much
more dramatically by Mn2+ and, intriguingly, displays
stereospecificity for the D-form Dopa/tyrosine enantiomers
(13). As discussed before, our recent kinetic studies (15) suggested
that Dopa and tyrosine form complexes with Mn2+ (the pH
dependence of the reaction forming the complex may explain the
different pH optimum for the Dopa/tyrosine-sulfating activity compared
with that for the dopamine sulfation, which does not involve such a
complex) that serve either as the obligatory (in the case of tyrosine)
or as a better (in the case of Dopa) substrate. The log K for
the formation of the tyrosine-Mn2+ complex is 1.5, whereas
that for the tyrosine-Mn2+-tyrosine complex is 5.0 (30,
31). Mn2+ coordinates not only with the carboxyl group of
the substrates (to form the complexes) but also with amino acid
residues on the enzyme (on formation of the enzyme-substrate complex).
Our previous studies using chimeric and point-mutated SULT1A3/SULT1A1s
had revealed that two variable regions (cf. Fig.
1A), and in particular residues 86, 89, and 146 therein, are important in determining the
distinct substrate specificity of these two otherwise highly homologous
enzymes. The current study aimed to elucidate further the structural
determinants for the unique stereoselectivity and Mn2+
dependence of the Dopa/tyrosine-sulfating activity of SULT1A3.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-thiogalactopyranoside, thrombin, and PAPS
were from Sigma. QuikChange site-directed mutagenesis kit and
XL1-Blue Epicurian coli competent cells were purchased from
Stratagene. Oligonucleotide primers were synthesized by MWG Biotech.
pGEX-2TK glutathione S-transferase gene fusion vector, E. coli BL21 host cells, and glutathione-Sepharose were
products of Amersham Biosciences. Carrier-free sodium
[35S]sulfate and Ecolume liquid scintillation fluid were
from ICN Biomedicals. Recombinant human bifunctional ATP
sulfurylase/adenosine 5'-phosphosulfate kinase was prepared as
described previously (22). Cellulose thin-layer chromatography (TLC)
plates were from EM Science. All other chemicals were of the highest
grades available commercially.
-D-thiogalactopyranoside overnight at room temperature, were collected by centrifugation and
processed for the purification of recombinant deletional SULT1A3 mutant
enzyme using the same procedure developed previously for the wild-type
SULT1A3 (7).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (61K):
[in a new window]
Fig. 1.
A, amino acid sequence comparison of
SULT1A3 and SULT1A1. Identical amino acid residues are
boxed. B, schematic representation of the
molecular architecture of wild-type SULT1A3 (labeled MMMM),
SULT1A1 (labeled PPPP), and chimeras.
Differential Roles of Variable Regions I and II of SULT1A3 in the Stereospecific Dopa/Tyrosine Sulfation and Its Stimulation by Mn2+-- This was studied by testing wild-type SULT1A3 and SULT1A1 and chimeric SULT1A3/SULT1A1s (cf. Fig. 1B) for their sulfation activities toward the Dopa and tyrosine enantiomers, in the presence or absence of Mn2+. Our previous kinetic studies (15) had confirmed that Mn2+ could stimulate dramatically the sulfation activity of wild-type SULT1A3 toward the tyrosine enantiomers. Mn2+, when added at a 5 mM concentration to the assay mixture, stimulated the sulfation activity of SULT1A3 toward D-tyrosine by two orders of magnitude in comparison with the basal activity determined without Mn2+, whereas the activity toward L-tyrosine was stimulated about ten times (15). The same assay conditions were used to evaluate the stimulatory effect of Mn2+ on the sulfation activities of wild-type SULT1A3 and SULT1A1 and their various chimeras. The activity data obtained (Table I) confirmed that the Mn2+-stimulated activity with D-tyrosine as substrate for wild-type SULT1A3 was about 135 times the basal activity. When L-tyrosine was used as substrate, the stimulation was 14-fold. Therefore, as in our previous findings, wild-type SULT1A3 (designated MMMM for comparison with the chimeras; cf. Fig. 1B) showed a distinct preference for the D-form enantiomer of tyrosine relative to the L-enantiomer. Wild-type SULT1A1 (PPPP), on the other hand, showed no sulfation activity toward either tyrosine enantiomer, with or without Mn2+. The data in Table I were analyzed to pinpoint which of the four regions (the two flanking regions and the middle variable Regions I and II) in the SULT1A3 molecule are required, respectively, for the basal tyrosine-sulfating activity, the stereospecificity, and the Mn2+ stimulation. Interestingly, the PMMP chimera displayed essentially the same level of basal activity, extent of stimulation by Mn2+, and stereospecificity as wild-type SULT1A3 (MMMM), whereas wild-type SULT1A1 (PPPP) showed none of these effects. These results indicated that the variable Regions I and II of the SULT1A3 can fully account for the tyrosine-sulfating activity and Mn2+ stimulation effect. Data on the chimeras with the variable Region II derived from SULT1A1 (MPPM, MMPM, and PMPP) showed that they were all incapable of catalyzing the sulfation of tyrosine enantiomers, indicating an absolute requirement for the variable Region II of the SULT1A3 molecule for the basal tyrosine-sulfating activity. In contrast, the chimeras MPMM and PPMP showed a small basal activity with D-tyrosine as substrate but without any Mn2+ stimulation effect. With L-tyrosine as substrate, however, no activity was detected with either of these two chimeras. These latter results suggested indirectly that the variable Region I of the SULT1A3 molecule, although not absolutely required for, may contribute substantially to its D-tyrosine sulfation activity. Moreover, the data indicated that this variable Region I is essential for the Mn2+ stimulation effect with D-tyrosine as substrate.
|
A limitation in the use of tyrosine as substrate is that the basal activities detected for the wild-type and chimeric SULTs were all quite low. Although the above-mentioned data indicated that the SULT1A3 Region I is required for the Mn2+ stimulation effect, it was difficult to determine whether it is truly required for the stereospecificity of SULT1A3 for the D-form tyrosine enantiomer. To better address this issue, Dopa enantiomers were used as substrates. Our previous kinetic studies (15) had demonstrated that wild-type SULT1A3 displayed significantly higher basal sulfation activities toward Dopa. Although much less dramatic compared with the sulfation of tyrosine, a 2- to 3-fold stimulation (at the Dopa concentrations used) of the Dopa-sulfating activity was observed in the presence of Mn2+. Therefore, using Dopa as substrate may prove to be more useful in investigating the structural requirements for the stereospecificity and Mn2+ stimulation effect. The sulfation activities of wild-type SULT1A3 and SULT1A1 and the chimeras toward D- or L-Dopa, in the presence or absence of 2.5 mM Mn2+, were determined. The results obtained are compiled in Table II. Wild-type SULT1A3 showed a distinct preference for D-Dopa. The Mn2+-stimulated activity of wild-type SULT1A3 with D-Dopa as substrate was 1.8 times the basal activity, whereas with L-Dopa it was almost 2-fold. The data obtained with the chimeras basically reinforce the conclusions reached using tyrosine as substrate. Moreover, the chimeras with the variable Region I derived from SULT1A1 and Region II from SULT1A3 (i.e. PPMP and MPMM), while exhibiting substantial basal sulfation activities toward both D- and L-Dopa, showed a preference for the D-enantiomer, especially with the PPMP chimera where the activities were higher. There appeared to be no significant Mn2+ stimulation effects with these chimeras. Because the absence of SULT1A3 variable Region I abolished the Mn2+ stimulation effect it follows that this Region I is required for the Mn2+ stimulation effect. The stereospecificity for the D-enantiomer, however, is present regardless of whether Region I is from the SULT1A3 or SULT1A1. The variable Region II of SULT1A3 may be required for its stereospecificity for the D-enantiomers. Moreover, the variable Region II of SULT1A3 is absolutely required for the basal Dopa/tyrosine-sulfating activity, whereas this variable Region I, although not absolutely required, substantially enhances this sulfation activity.
|
Identification of Specific Amino Acid Residues Required for the Mn2+ Stimulation of the Sulfation of Tyrosine and Dopa Enantiomers-- To investigate further the structural determinants for the Mn2+ stimulation of the sulfation of tyrosine and Dopa enantiomers, point-mutated SULT1A3s, targeted at specific amino acid residues within the variable Regions I and II, were tested. By employing the site-directed mutagenesis technique, we had generated, expressed, and purified nine point-mutated SULT1A3s (five single mutants, three double mutants, and a triple mutant) targeted at amino acid residues in the two variable regions (8). One other single mutant involved a conserved residue Lys-48 that most likely participates in catalysis (8). The sulfation activities of these point-mutated SULT1A3s toward D- or L-tyrosine, in the presence or absence of Mn2+, were determined.
Fig. 2 shows a bar graph plotted based on
the results obtained in experiments in which the
Mn2+-stimulated D-tyrosine-sulfating activities
of wild-type and mutant SULT1A3s were compared with their basal
activities. In the absence of Mn2+, only a small sulfation
activity was detected in all cases. It was evident that the
Mn2+-stimulated D-tyrosine-sulfating activity
found with the wild-type enzyme (135 times the basal activity) was
largely retained in the N85K, E89I, and H143Y mutants, as well as in
the N85K/E89I double mutant. This indicates that amino acid residues
Asn-85, Glu-89, and His-143 are not required for the
Mn2+-stimulated sulfation activity toward
D-tyrosine, because their mutation did not abolish the
stimulation by Mn2+. In contrast, both the E146A and D86A
mutants and the double or triple mutants involving one or both of these
two residues (i.e. N85K/D86A, D86A/E89I, and
D86A/E89I/E146A) showed very low sulfation activities toward
D-tyrosine, with or without Mn2+. Mutation at
residue Lys-48 resulted in the virtual abolishment of the sulfation
activity toward D-tyrosine, with or without
Mn2+. As revealed in our previous studies (8), however,
this may be because Lys-48 is a catalytic residue (conserved in SULT1A3 and SULT1A1, as well as other cytosolic STs), and its mutation would
result in the loss of virtually the entire sulfation activity.
|
Fig. 3 shows a bar graph plotted based on
the results obtained in experiments in which the
Mn2+-stimulated L-tyrosine-sulfating activities
of wild-type and mutant SULT1A3s were compared with their basal
activities. The results were in general similar to those obtained with
D-tyrosine as substrate, though the extent of stimulation
by Mn2+ was considerably lower in all cases. One exception,
however, is that the E146A mutant exhibited a considerable stimulation by Mn2+ of its sulfation activity toward
L-tyrosine, albeit at lower activity levels. This was in
fact also the case in its sulfation activity toward
D-tyrosine, which showed a four times stimulation by
Mn2+ (this Mn2+ stimulation effect in Fig. 2,
however, is overshadowed by the much more dramatic Mn2+
stimulation effects observed with wild-type SULT1A3 and N85K, E89I,
H143Y, and N85K/E89I mutant clones). These latter results therefore
indicate that the Glu-146 residue is not pivotal for the
Mn2+ stimulation of the sulfation of the tyrosine
enantiomers by SULT1A3.
|
To summarize, the data in Figs. 2 and 3 appear to rule out the requirement for residues Asn-85, Glu-89, Glu-146, and His-143 in the Mn2+ stimulation of the tyrosine-sulfating activity of SULT1A3. The role of residue Asp-86 is not as clear. This is because the sulfation activities toward both D- and L-tyrosine, either in the presence or absence of Mn2+, were very low for the D86A mutant, and the data were somewhat ambiguous. Whether the lowered sulfation activities were the result of a drastic loss of the basal tyrosine-sulfating activity (as appeared to be the case with the E146A mutant) or a loss of the Mn2+ stimulation effect or both was not clear. To resolve this issue, we carried out assays using the same set of wild-type and chimeric SULT1A3/SULT1A1s (cf. Fig. 1) with D- and L-Dopa as substrates. As revealed in Table II, wild-type SULT1A3 showed a much higher basal activity toward Dopa, as well as a clearly detectable stimulation by Mn2+ (though not as dramatic as in the case of tyrosine). Therefore, using D- and L-Dopa as substrates, it may be possible to uncouple the Dopa/tyrosine-sulfating activity from its stimulation by Mn2+.
Fig. 4 shows a bar graph plotted based on
the results obtained in experiments in which the
Mn2+-stimulated D-Dopa-sulfating activities of
wild-type and mutant SULT1A3s were compared with their basal
activities. In contrast to the case with tyrosine as substrate,
considerably higher basal activities for wild-type and mutant SULT1A3s
were observed even in the absence of Mn2+. It is evident
from the figure that the Mn2+-stimulated
D-Dopa-sulfating activity associated with the wild-type enzyme was essentially retained in the N85K, E89I, and H143Y mutants, as well as in the N85K/E89I double mutant. It is also clear that for
the D86A mutant and the double (N85K/D86A and D86A/E89I) and triple
(D86A/E89I/E146A) mutants involving D86A mutation, there was no longer
noticeable Mn2+ stimulation of the basal activities toward
D-Dopa. Besides reinforcing our earlier conclusion with
regard to residues Asn-85, Glu-89, His-143, and Glu-146 not being
required for the Mn2+ stimulation effect, these results
unequivocally confirm the primary requirement for residue Asp-86 in the
Mn2+ stimulation of the Dopa/tyrosine-sulfating activity of
SULT1A3. Because residue Asp-86 is present within the variable Region
I, the above-mentioned results are in line with the earlier conclusion that the presence of the variable Region I from SULT1A3 is required for
the Mn2+ stimulation of the Dopa/tyrosine-sulfating
activity (cf. above-mentioned studies using the chimeras).
As a negatively charged residue, Asp-86 is inherently capable of
mediating the binding of Mn2+ to SULT1A3.
|
Identification of Specific Amino Acid Residues Required for the
Stereospecificity of the Sulfation of Tyrosine and Dopa
Enantiomers--
Fig. 5 shows a bar
graph plotted based on the data in which the
D-tyrosine-sulfating activities of wild-type and mutant
SULT1A3s were compared with their L-tyrosine-sulfating
activities (both in the presence of Mn2+). It is clear from
the figure that wild-type SULT1A3 and N85K, E89I, N85K/E89I, and H143Y
mutants exhibited stereospecificity for the D-enantiomer of
tyrosine. No clearly discernable stereospecificity was observed for
E146A, D86A, and double or triple mutants involving one or both of
these two mutations. This is likely because of the inherently low
sulfation activities of these mutants toward tyrosine, which would not
allow for a clear-cut distinction of the substrate preference for
D- or L-enantiomer. To better resolve the
problem, the data on the comparison between the
D-Dopa-sulfating activities of wild-type and mutant
SULT1A3s and their L-Dopa-sulfating activities (both in the
presence of Mn2+) were analyzed. From the bar graph plotted
(Fig. 6), the preference for
D-Dopa over L-Dopa was clearly found for the
D86A mutant and the two double mutants involving D86A mutation. Based
on these results, it can be concluded that residue Asp-86 is not
required (see also the following studies on deletional mutants) for the stereospecificity of SULT1A3, at least when Mn2+
stimulation and binding to this residue is not involved. In the case of
the E146A mutant, its low level of activity toward Dopa enantiomers
still would not allow for a clear distinction regarding stereospecificity. However, when the data for the E146A mutant were
collated and plotted together (Fig. 7),
the E146A mutant no longer displayed the specificity for the
D-enantiomer of tyrosine or Dopa. It is therefore clear
that residue Glu-146 is the one that primarily directs the
stereospecificity of SULT1A3 for the D-enantiomer of Dopa
or tyrosine. This is in accordance with our earlier conclusion from the
studies using chimeras that the SULT1A3 variable Region II is the one
responsible for the stereospecificity of the Dopa/tyrosine-sulfating
activity of SULT1A3.
|
|
|
Sulfation Activities of the Residues 84-86 and 84-90 Deletional SULT1A3 Mutants toward Dopa and Tyrosine: Stereospecificity and Stimulatory Effect of Mn2+-- The above-mentioned studies have pinpointed the residue Glu-146 of SULT1A3 as being primarily responsible for the stereospecificity for the D-enantiomers of Dopa and tyrosine. To further clarify these points, the activities of two deletional SULT1A3 mutants (lacking, respectively, residues 84-86 and residues 84-90) toward tyrosine (5 mM final concentration) and Dopa (1 mM final concentration) enantiomers were measured in the absence of Mn2+. The residues 84-86 deletional mutant exhibited no detectable activities toward any of these substrates (or with dopamine). The residues 84-90 deletional mutant displayed detectable activity though this was much lower, as compared with that of the wild-type SULT1A3. Surprisingly, this deletional mutant seemed to have completely lost the stereospecificity for the D-enantiomers of tyrosine or Dopa as is clear from the data compiled in Table III. This seems to suggest a role for the "mobile loop" (encompassing residues 84-92; see Ref. 17 and below) per se as a steric selector, in the absence of Mn2+, for the D-enantiomers of these substrates.
|
Proposed Model Explaining the Stimulatory Effect of Mn2+ and the Stereoselectivity for D-Enantiomers of Dopa/Tyrosine-- SULT1A3 is known to be present as a homodimer in its native state (14). Previous x-ray crystallography study (17) had shown that, in the crystal structure, residues 84-92 corresponding to the variable Region I (cf. Fig. 1A) from one monomer form a mobile loop that intercalates into the active site of the other monomer and may block the proper positioning of certain acceptor substrates. In addition to this mobile loop, the active site is also guarded by key residues (particularly Glu-146) (10, 11) from the variable Region II of the same monomer. This model had been used to explain our previous kinetic studies (15) by suggesting that the mobile loop might not hinder the positioning of the physiological substrate, dopamine, whose positively charged amino group may be stabilized by interaction with the negatively charged Glu-146 residue (8, 10, 11). It was proposed that the variable Region I loop may, however, act to prevent the positioning of xenobiotic substrates (represented by D-Dopa or D-tyrosine) that carry, for example, an extra carboxyl group, as compared with dopamine. In this model, Mn2+ exerts its stimulatory effect by complexing with the negatively charged carboxyl group of Dopa or tyrosine and forming a bridge with a residue in the loop. This would peg back the loop, thus allowing the proper positioning of these substrates.
The essential features of this model could also be used to explain the results obtained in the current study. However, one important modification has to be made. The previously reported x-ray structure of SULT1A3 (17, 32) demonstrated that the interaction between the subunits of the homodimer, in the crystal structure, takes place at the surface near the substrate binding sites of the subunits, which is consistent with the Region I loop from one monomer intercalating into the substrate binding site of the other monomer. This idea seemed to be supported by buried surface area calculations and data on the x-ray structure of other SULT enzymes (33). However, more recent work using cross-linking and limited proteolysis, in conjunction with mass spectrometry (34), suggested that the physiological dimer interface in solution (as opposed to the crystal dimer interface) may be near the C-terminal region of the subunit, away from the surface of the substrate binding site. Site-directed mutagenesis, along with gel filtration data, pinpointed a stretch of 10 amino acid residues near the C-terminal end of the subunit (in SULT1A3 this occurs in the region between Lys-265 and Glu-274) with the sequence KXXXTVXXXE, conserved in all the dimeric SULTs, dubbed the "dimerization motif." This dimerization motif forms a hydrophobic "zipper" with ion pairs at the ends, at the interface between the dimers (34). This finding basically ruled out the possibility of the variable Region I mobile loop, which lies at the other end of the subunits, intercalating into the active site of the other subunit in solution. But because the evidence from our laboratory and others (see Refs. 7-10 and 15 and the present study) implicates the residues 84-89 in the variable Region I of SULT1A3 in interactions with the acceptor substrate at the active site, and because the x-ray crystallographic evidence indicates that residues 84-92 of SULT1A3 form a mobile loop, we must conclude that this loop, in solution, rather than intercalating into the substrate binding site of the other subunit of the dimer, by the same free energy minimization criteria, tucks back into the substrate binding site of the same subunit. This loop formed by the variable Region I therefore would still exert the same effects, albeit in the same subunit.
This model suggests that the loop, although not hindering the entry of the physiological substrate dopamine, may block the entry of other substrates such as Dopa and tyrosine with an extra carboxyl group. Mn2+ stimulates the activity with these latter substrates by complexing on the one hand with the carboxyl group of these substrates and on the other hand specifically with the Asp-86 residue, thus pegging back the loop. This model explains why the D86A SULT1A3 mutant failed to show any Mn2+-stimulated sulfation activity with D-Dopa as substrate (cf. Fig. 4). Data from our studies on the kinetics of the sulfation of dopamine and of the tyrosine and Dopa enantiomers by SULT1A3 and the effects of Mn2+ on the kinetics of sulfation seem to support this model (15). The interaction of SULT1A3 with dopamine appears to follow typical Michaelis-Menten-type kinetics, and Mn2+ seems to exert a smaller stimulatory effect (increasing Vmax by 2-3-fold but increasing Km only slightly) by binding directly to the Asp-86 residue of SULT1A3, and no complex with dopamine seems to be involved (15). In contrast, the interaction of SULT1A3 with D-tyrosine and D-Dopa involves a tyrosine-Mn2+ or a tyrosine-Mn2+-tyrosine complex as an obligate substrate and a Dopa-Mn2+ or a Dopa-Mn2+-Dopa complex as a better substrate than Dopa (15). With these substrates, Mn2+ appears to form a bridge between the carboxyl group of the substrate with which it is complexed and the Asp-86 residue of the enzyme molecule.
The stereospecificity of SULT1A3 for the D-enantiomers of Dopa and tyrosine and the importance of the Glu-146 residue in this effect will require more detailed modeling studies and perhaps quantitative structure activity relationship analysis (17). However, it is clear that the interaction of the negatively charged Glu-146 residue with the positively charged amino groups of Dopa and tyrosine is an important factor in the stereospecific interaction between SULT1A3 and these substrates (8, 10, 11). Our studies with the 84-90 deletional mutant also revealed that, in the absence of Mn2+, the presence of the variable Region I loop as a whole may serve as an important steric selector, because, in its absence, the stereoselectivity of the SULT1A3 for the D-enantiomers of Dopa and tyrosine appears to be lost. Our previous studies with dopamine as substrate (8) and the present study with Dopa and tyrosine as substrates revealed that besides the E146A mutant, the D86A and E89I mutants also showed reduced basal activities toward these substrates. In the case of the E146A mutant, the loss of activity could be because of the loss of an energetically favorable electrostatic interaction. With the D86A and E89I mutants, although the mechanism remains unclear, it is possible that the lower basal activity could result from altered structural interactions making the variable Region I loop even more restrictive. Some support for this possibility comes from our recent studies on the extent of inhibition of the activity of SULT1A3 and its mutants by 2,6-dichloro-4-nitrophenol (DCNP).2
Our model suggests that SULT1A3, under normal circumstances, acts only on its physiological substrate dopamine, thereby regulating its levels. This makes sense from the viewpoint of cellular economy, because sulfation is an energetically expensive process that uses PAPS, the synthesis of one molecule of which requires the expenditure of three high energy phosphate bonds of ATP (3). Under conditions of oxidative stress (which may result in the release of Mn2+ from mitochondria into the cytosol), however, the constraining loop in the active site may be pinned back by a substrate-Mn2+ complex, allowing the proper positioning, and therefore the sulfation, of non-physiological xenobiotic molecules present at higher levels (15). It is possible that other candidate molecules serving as substrates in this way may form complexes with Mn2+ with much higher log K values.
The physiological significance, if any, of the Mn2+
stimulation of the Dopa/tyrosine-sulfating activity of SULT1A3, and the specificity for the D-enantiomers of these substrates,
which has also been demonstrated in cell culture and in cell-free
extracts (13, 29), is somewhat speculative and has been considered in a
previous report (15). From a more practical perspective, however, the
stimulation of the detoxifying activity of SULT1A3 by Mn2+
and its stereoselective action may have implications for the detoxifying activity of the SULTs toward chiral drugs (18-21). We are
currently investigating the stereoselective action of SULT1A3 and its
mutants on some chiral dopamine analogs widely used as drugs.3 Besides throwing
further light on the structure-function relationships we have
considered here, the principles emerging may enable some engineering of
the protein with regard to its stereospecific action.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the American Heart Association (Texas Affiliate) (to M. C. L.), a UTHCT President's Council Research Membership Seed Grant (to M. C. L.), and an award from the Naito Foundation (to M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Biomedical Research
Center, The University of Texas Health Center, 11937 U. S. Highway
271, Tyler, TX 75708. Tel.: 903-877-2862; Fax: 903-877-2863; E-mail:
ming.liu@uthct.edu.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M203108200
2 T. Sugahara, I. Oxendine, T. G. Pai, and M.-C. Liu, unpublished data.
3 M. C. Liu, I. Oxendine, and T. G. Pai, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ST, sulfotransferase; PAPS, adenosine 3'-phosphate,5'-phosphosulfate; Dopa, 3,4-dihydroxyphenylalanine; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; TLC, thin-layer chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Mulder, G. J., and Jakoby, W. B. (1990) in Conjugation Reactions in Drug Metabolism (Mulder, G. J. , and Jakoby, W., eds) , pp. 107-161, Taylor and Francis, Ltd., London |
2. | Falany, C., and Roth, J. A. (1993) in Human Drug Metabolism; From Molecular Biology to Man (Jeffery, E. H., ed) , pp. 101-115, CRC Press, Inc., Boca Raton, FL |
3. | Lipmann, F. (1958) Science 128, 575-580 |
4. |
Weinshilboum, R. M.,
Otterness, D. M.,
Aksoy, I. A.,
Wood, T. C.,
Her, C. T.,
and Raftogianis, R. B.
(1997)
FASEB J.
11,
3-14 |
5. | Coughtrie, M. W. H., Sharp, S., Maxwell, K., and Innes, N. P. (1998) Chem. Biol. Interact. 109, 3-27[CrossRef][Medline] [Order article via Infotrieve] |
6. | Duffel, M. W. (1997) in Comprehensive Toxicology (Guengerich, F. P., ed) , pp. 365-383, Elsevier Science, Ltd., Oxford |
7. |
Sakakibara, Y.,
Takami, Y.,
Nakayama, T.,
Suiko, M.,
and Liu, M.-C.
(1998)
J. Biol. Chem.
273,
6242-6247 |
8. |
Liu, M.-C.,
Suiko, M.,
and Sakakibara, Y.
(2000)
J. Biol. Chem.
275,
13460-13464 |
9. | Brix, L. A., Duggleby, R. G., Gaedigk, A., and McManus, M. E. (1999) Biochem. J. 337, 337-343[CrossRef][Medline] [Order article via Infotrieve] |
10. | Brix, L. A., Barnett, A. C., Duggleby, R. G., Leggett, B., and McManus, M. E. (1999) Biochemistry 38, 10474-10479[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Dajani, R.,
Hood, A. M.,
and Coughtrie, M. W. H.
(1998)
Mol. Pharmacol.
54,
942-948 |
12. | Rubin, G. L., Sharp, S., Jones, A. L., Glatt, H., Mills, J. A., and Coughtrie, M. W. H. (1996) Xenobiotica 26, 1113-1119[Medline] [Order article via Infotrieve] |
13. | Sakakibara, Y., Katafuchi, J., Takami, Y., Nakayama, T., Suiko, M., Nakajima, H., and Liu, M.-C. (1997) Biochim. Biophys. Acta 1355, 102-106[Medline] [Order article via Infotrieve] |
14. | Heroux, J. A., and Roth, J. A. (1988) Mol. Pharmacol. 34, 194-199[Abstract] |
15. |
Pai, T. G.,
Ohkimoto, K.,
Sakakibara, Y.,
Suiko, M.,
Sugahara, T.,
and Liu, M.-C.
(2002)
J. Biol. Chem.
277,
43813-43820 |
16. | Suiko, M., Sakakibara, Y., Awan-Khan, R., Sakaida, H., Yoshikawa, H., Ranasinghe, J. G. S., and Liu, M.-C. (1998) J. Biochem. 124, 707-711[Abstract] |
17. |
Dajani, R.,
Cleasby, A.,
Neu, M.,
Wonacott, A. J.,
Jhoti, H.,
Hood, A. M.,
Modi, S.,
Hersey, A.,
Taskinen, J.,
Cooke, R. M.,
Manchee, G. R.,
and Coughtrie, M. W. H.
(1999)
J. Biol. Chem.
274,
37862-37868 |
18. | Wilson, A. A., Wang, J., Koch, P., and Walle, T. (1997) Xenobiotica 27, 1147-1154[CrossRef][Medline] [Order article via Infotrieve] |
19. | Walle, U. K., Persola, G. R., and Walle, T. (1993) Br. J. Clin. Pharmacol. 35, 413-418[Medline] [Order article via Infotrieve] |
20. | Persola, G. R., and Walle, T. (1993) Chirality 5, 602-609[Medline] [Order article via Infotrieve] |
21. | Walle, T., and Walle, U. K. (1992) Drug Metab. Dispos. 20, 333-336[Medline] [Order article via Infotrieve] |
22. | Yanagisawa, K., Sakakibara, Y., Suiko, M., Takami, Y., Nakayama, T., Nakajima, H., Takayanagi, K., Natori, Y., and Liu, M.-C. (1998) Biosci. Biotechnol. Biochem. 62, 1037-1040[Medline] [Order article via Infotrieve] |
23. | Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract] |
24. | Liu, M.-C., and Lipmann, F. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3695-3698[Abstract] |
25. | Fernando, P. H. P., Karakawa, A., Sakakibara, Y., Ibuki, H., Nakajima, H., Liu, M.-C., and Suiko, M. (1993) Biosci. Biotechnol. Biochem. 5, 1974-1975 |
26. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
27. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
28. | Butler, P. R., Anderson, R. J., and Venton, D. L. (1983) J. Neurochem. 41, 630-639[Medline] [Order article via Infotrieve] |
29. | Suiko, M., Sakakibara, Y., Nakajima, H., Sakaida, H., and Liu, M.-C. (1996) Biochem. J. 314, 151-158[Medline] [Order article via Infotrieve] |
30. | Martell, A. E., and Smith, R. M. (1974) Critical Stability Constants , Vol. 1 , Plenum Press, New York |
31. | Gergely, A., Nagypal, I., and Kiraly, B. (1971) Acta Chim. Acad. Sci. Hung. 68, 285 |
32. | Bidwell, L. M., McManus, M. E., Gaedigk, A., Kakuta, Y., Negishi, M., Pedersen, L. C., and Martin, J. F. (1999) J. Mol. Biol. 293, 521-530[CrossRef][Medline] [Order article via Infotrieve] |
33. | Pedersen, L. C., Petrotchenko, E. V., and Negishi, M. (2000) FEBS Lett. 475, 61-64[CrossRef][Medline] [Order article via Infotrieve] |
34. | Petrotchenko, E. V., Pedersen, L. C., Borchers, C. H., Tomer, K. B., and Negishi, M. (2001) FEBS Lett. 490, 39-43[CrossRef][Medline] [Order article via Infotrieve] |