Steroid Sulfotransferase Activity and X-ray Analysis
315
TABLE 1. Km and Vmax values for sulfonation of E , DHEA, and DES
2
E2
DHEA
DES
Km (M)
Vmax (pmol/nmol/min)
Km (M)
Vmax (pmol/nmol/min)
Km (M)
Vmax (pmol/nmol/min)
EST
HST
PST
0.28 Ϯ 0.13
7.9 Ϯ 3
174 Ϯ 17
216 Ϯ 24
34 Ϯ 13
1.1 Ϯ 0.2
52 Ϯ 27
421 Ϯ 48
0.08 Ϯ 0.02
0.13 Ϯ 0.03
0.46 Ϯ 0.15
34 Ϯ 5
53 Ϯ 5
37 Ϯ 4
Reaction mixtures contained 2 and 180 g of bacterially expressed EST for E
0
3
2
and DHEA sulfotransferase activities, respectively. Substrate concentrations used were 0.088, 0.176,
.264, 0.352, 0.44, 0.88, and 1.32 M of E2 and 0.56, 1.12, 2.24, 4.48, 5.6, 15.0, and 30.0 M of DHEA. HST (34 and 3.4 g, respectively) was incubated with E2 (5.0, 10.0,
0.0, and 50.0 M) and DHEA (0.25, 0.5, 0.75, 1.0, and 2.0 M), respectively. The DES sulfotransferase activities were measured for EST (1.5 g), HST (1.5 g), and PST
(5 g). DES concentrations varied from 0.02, 0.033, 0.05, 0.08, 0.10, 0.20, to 0.40 M when EST or HST was used as enzyme. For PST, DES concentrations used were 0.1, 0.2,
0
.5, 0.8, 1.0, and 2.0 M. Other assay conditions were described in “Materials and Methods.” A non-linear regression program (created in-house and based on a program given
in Ref. 13) was employed to calculate Km and Vmax values.
Specificity for Sulfonation Activity of
Endogenous Steroids
indicate that DES sulfotransferase activity of both EST and
HST is stereospecific to the trans-isomer.
We have shown in a previous paper that the bacterially
expressed mouse EST exhibits extremely high E2 and
Kinetics of DES Sulfotransferase Activity
estrone (E ) sulfotransferase activities compared with
1
DHEA, progesterone, and testosterone sulfotransferase ac-
tivities [11]. Consistent with this previous finding, the Km
To further compare the DES sulfotransferase activities of
EST, HST, and PST, we measured their Km and Vmax
values. A typical Michaelis–Menten kinetics plot is shown
in Fig. 3, and these values are summarized in Table 1. The
Km value (0.08 M) of EST for DES sulfotransferase
activity was 3.5-fold lower than that of the corresponding
E2 sulfotransferase activity. Surprisingly, HST efficiently
catalyzed DES sulfotransferase activity: the Km value for
DES sulfotransferase activity was 10-fold lower than that
for the original DHEA sulfotransferase activity. The
Vmax/Km ratios (405 and 382, respectively) showed that
HST sulfated DES and DHEA with the same turnover
rates. These rates were very similar to the ratio (425) of
EST for DES sulfotransferase activity. PST also sulfated
DES, although its Vmax/Km ratio for DES sulfotransferase
activity was Ͼ 5-fold higher than those for EST and HST.
DES sulfonation has also been reported with human PST
and EST [14]. Consistent with our present findings, the
human enzymes exhibit a much poorer substrate specificity
value of EST for E sulfotransferase activity was 120-fold
2
lower than that for the corresponding DHEA sulfotrans-
ferase activity, while the Vmax for E was 3-fold higher than
2
that of DHEA (Table 1). Conversely, for DHEA, HST
displayed a 7-fold lower K and a 2-fold higher V
value
m
max
than for E sulfotransferase activities. EST and HST are
2
specific in sulfating E and DHEA, respectively, although
2
the degree of substrate specificity appears to be greater in
the former. The absolute Km values of the mouse enzymes
were somewhat different from those reported previously for
the corresponding EST and HST in humans and guinea
pigs [14, 15]. Consistently, however, their relative substrate
specificities toward E2 and DHEA were in very good
agreement.
Metabolism of DES by Sulfotransferases
DES metabolites formed by the sulfotransferases were sub-
jected to HPLC analysis (Fig. 2). Radioactive DES used as
substrate contained both trans- and cis-isomers that were
separated by HPLC. No nonenzymatic product was formed
from DES even in the presence of the co-factor PAPS (Fig.
toward DES sulfonation than toward E sulfonation. The
2
reported absolute K values differ significantly between the
m
human and mouse ESTs, which remains to be investigated
in the future. However, it is not surprising to observe these
differences, since substrate specificity of enzymes that me-
tabolize xenobiotics (e.g. cytochrome P450) can be altered
by only a minor amino acid substitution [16].
2
A). A short incubation of DES with EST decreased
specifically the peak height of the trans-isomer and two
peaks of DES metabolites increased (Fig. 2B). The fast-
eluting peak was co-chromatographed with chemically
synthesized DES disulfate ester, while the late-eluting peak
had the same retention time as DES monosulfate ester.
Monosulfate ester appeared to be formed first, since more of
this intermediate was produced than the disulfate ester in
the 6-min incubation (Fig. 2B), while only the disulfate
ester appeared in the 25-min incubation (Fig. 2C). HST
also sulfated DES first to monosulfate and then to disulfate
esters (data not shown). Interestingly, the cis-isomer of DES
remained unsulfated even in the 25-min incubation with
either sulfotransferase (Fig. 2C). These results, therefore,
EST Crystals
Crystals of EST diffracted X-rays to a resolution of 2.5 Å.
(Fig. 4). Redundancy and completeness were 5.5 and
97.5%, respectively. The space group was P2 2 2 with unit
1
1
cell dimensions a ϭ 96.4 Å, b ϭ 80.4 Å, and c ϭ 80.8 Å.
There were two molecules in the asymmetric unit giving a
3
V of 2.24 Å /Da, which corresponded to a solvent content
M
of 46% [17]. A search for heavy atom derivatives is
currently in progress.