(Received for publication, April 10, 1995; and in revised form, June 21, 1995)
From the
3-Hydroxysteroid dehydrogenase (3
-HSD) and steroid
-isomerase were copurified as a single protein from human
placental microsomes. Because NADH is an essential activator of
isomerase (K
= 2.4 µM, V
= 0.6 µmol/min/mg), the affinity
alkylating nucleotide,
8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5`-diphosphate
(8-BDB-TADP), was synthesized. 8-BDB-TADP activates isomerase (K
= 338 µM, V
= 2.1 µmol/min/mg) prior to
inactivating the enzyme. The inactivation kinetics for isomerase fit
the Kitz and Wilson model for time-dependent, irreversible inhibition
by 8-BDB-TADP (K
= 314
µM, first order maximal rate constant k
= 7.8
10
s
).
NADH (50 µM) significantly protects isomerase from
inactivation by 8-BDB-TADP (100 µM). The isomerase
activity is inactivated more rapidly by 8-BDB-TADP as the concentration
of the affinity alkylator increases from 67 µM (t
= 8.4 min) to 500 µM (t
= 2.4 min). In sharp contrast,
the 3
-HSD activity is inactivated more slowly as the concentration
of 8-BDB-TADP increases from 67 µM (t
= 4.8 min) to 500 µM (t
= 60.0 min). We hypothesized that the paradoxical kinetics
of 3
-HSD inactivation is a consequence of the activation of
isomerase by 8-BDB-TADP via a nucleotide-induced shift in enzyme
conformation. Biophysical support for an NADH-induced conformational
change was obtained using stopped-flow fluorescence spectroscopy. The
binding of NADH (10 µM) quenches the intrinsic
fluorescence of the enzyme protein in a time-dependent manner (rate
constant k
= 8.1
10
s
, t
= 85 s). A
time lag is also observed for the activation of isomerase by NADH. This
combination of affinity labeling and biophysical data using nucleotide
derivatives supports our model for the sequential reaction mechanism;
the cofactor product of the 3
-HSD reaction, NADH, activates
isomerase by inducing a conformational change in the single,
bifunctional enzyme protein.
3-Hydroxy-
-steroid dehydrogenase
(3
-HSD, (
)EC 1.1.1.145) and steroid
-isomerase
(EC 5.3.3.1) sequentially catalyze the two step-conversion of
3
-hydroxy-5-ene steroids to 3-keto-4-ene steroids on a single
enzyme protein purified from human placenta(1) , bovine
adrenals(2) , rat adrenals(3) , or rat
testis(4) . With pregnenolone shown as a representative
3
-hydroxy-5-ene substrate, the enzyme reactions are as follows:
This reaction scheme shows the reduction of NAD to NADH by the 3
-HSD activity and the requirement of
coenzyme (without further conversion) for the activation of
isomerase(1) . Because 3
-HSD and isomerase were
inactivated at different rates by 2
-bromoacetoxyprogesterone (5) and exhibited different types of inhibition by N,N-diethyl-4-methyl-3-oxo-4-aza-5
-androstene-17
-carboxamide (6) , it appeared that the two activities were catalyzed at
separate sites on the same enzyme.
Our studies of purified human
placental 3-HSD/isomerase have suggested that NADH mediates a
shift in enzyme activity from dehydrogenase to isomerase by inducing a
conformational change in the enzyme protein. NADH is the coenzyme
product of the 3
-HSD reaction and is a potent, essential activator
of isomerase activity(7) . NADH, but not NAD
,
completely protects both the 3
-HSD and isomerase activities from
inactivation by the substrate-site affinity alkylators,
2
-bromoacetoxyprogesterone (5) and
5,10-secoestr-4-yne-3,10,17-trione(8) . Finally, the
isomerase-site-directed secosteroid,
5,10-secoestr-4-yne-3,10,17-trione, inactivated the isomerase activity
with the expected first-order kinetics but inactivated the 3
-HSD
activity in an unexpected manner. As the concentration of the
alkylating secosteroid increased, the rate of 3
-HSD inactivation
paradoxically decreased (8) instead of increasing in accordance
with the Kitz and Wilson model of irreversible enzyme
inhibition(9) .
In this report, the interaction of
3-HSD/isomerase with coenzyme is studied using the cofactor-site
affinity alkylator, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine
5`-diphosphate (8-BDB-TADP). In addition, the time-dependent effects of
NADH binding on the intrinsic enzyme fluorescence and on isomerase
activation are measured. 8-BDB-TADP, as well as the 6- and
2-(4-bromo-2,3-dioxobutyl)thio-derivatives of ADP, have been employed
previously to study nucleotide binding by pyruvate kinase (10) and isocitrate dehydrogenase(11, 12) .
Demonstration that the binding of substrate triggered a time-dependent
quenching of protein fluorescence (13) or stimulation of enzyme
activity (14) provided evidence for a ligand-induced
conformational change in Escherichia coli dihydrofolate
reductase. The unprecendented use of both kinetic data from affinity
labeling and spectroscopic measurements of coenzyme binding furnishes a
definitive test of our hypothesis that NADH induces a conformational
change that is critical to the sequential reaction mechanism of
3
-HSD/isomerase.
The decomposition rate of 8-BDB-TADP in 0.03 M MES buffer, pH 7.0, was determined by the loss of bromide at 22 °C as previously described(11) . The half-time for bromide loss was 56 min, in agreement with the reported half-life of 50 min under similar conditions(10) .
In protection studies, the control and
experimental mixtures contained the same concentration of the
potentially protecting steroid or cofactor with no increase in final
solvent content compared to incubations without protector. The
concentrations of these ligands were at least three times the K or K
measured for 3
-HSD or isomerase activity to facilitate
competition with a subsaturating concentration of 8-BDB-TADP (100.0
µM).
In mixed nucleotide-activator
analysis(7) , the stimulation of isomerase activity was
measured at 241 nm using 5-pregnene-3,20-dione (12.0 µM)
as substrate and kinetically equivalent concentrations (0.5 K
) of NADH alone (1.2 µM),
8-BDB-TADP alone (170 µM), or a mixture of the two
activators (each at 0.5
K
).
Nonspecific alkylation by 8-BDB-TADP was evaluated by preincubating the enzyme with ethyl bromoacetate (100.0 µM) for 30 min followed by the addition of 8-BDB-TADP (100.0 µM) using the conditions described above for enzyme inactivation. The rates of isomerase inactivation by 8-BDB-TADP were compared with or without preincubation with ethyl bromoacetate.
Assays that monitored the
loss of 3-HSD or isomerase activity during enzyme inactivation
were performed on aliquots taken from the incubation mixtures at
appropriate time intervals in duplicate according to our published
conditions(5) . The slope of the initial linear increase in
absorbance at 340 nm (due to NADH production during the oxidation of
pregnenolone) per unit time was used to determine 3
-HSD activity.
Isomerase activity was measured by the initial absorbance increase at
241 nm (due to progesterone formation from 5-pregnene-3,20-dione) as a
function of time. Changes in absorbance were measured with a Varian
(Palo Alto, CA) Cary 219 recording spectrophotometer. Nonspecific and
spontaneous enzyme activity were determined using blanks that contained
either no steroid substrate or no enzyme, respectively. The incubation
conditions cited in this report minimized spontaneous activity in the
enzyme assays, and all measurements of enzyme activity were corrected
for any observable nonspecific conversion of substrate.
The stoichiometry and dissociation constant for the binding of NADH
to the enzyme were determined using a Proton Technology International
(South Brunswick, NJ) Alpha Scan 4000 spectrofluorometer. The decrease
in intrinsic enzyme fluorescence produced by titration with NADH
yielded a value for maximal fluorescence change at site saturation
(F
). After correction with a blank
tryptophan titration, each NADH concentration yielded a
F value used to obtain
=
F/
F
. The data was plotted as
1/(1-
) versus [NADH]/
according to
Stinson and Holbrook (17) to yield a stoichiometry and
dissociation constant for NADH binding. For the stoichiometry study,
the actual concentration of enzyme protein was determined by
ultraviolet spectral measurements in 6.0 N guanidine (34 Tyr
plus 16 Trp residues/enzyme dimer yielded a molar extinction
coefficient at 280 nm of 1.346
10
/dimer).
The
non-site-directed alkylator, ethyl bromoacetate (100.0 µM,
100/1 alkylator to enzyme molar ratio) was preincubated with the enzyme
for 30 min (with less than 10% inactivation), and then 8-BDB-TADP
(100.0 µM) was added to the same incubation mixture.
Isomerase activity was rapidly inactivated (t = 5.0 min) by 8-BDB-TADP at the same rate measured for
8-BDB-TADP (100.0 µM) without preincubation with ethyl
bromoacetate (data not shown). These observations are evidence that
8-BDB-TADP inactivates isomerase with good specificity.
Figure 1:
Inactivation of 3-HSD and
isomerase by 8-BDB-TADP. The 3
-HSD (panelA) or
isomerase (panelB) activity of the enzyme (1.0
µM) was inactivated by 67.0 µM (
),
100.0 µM (
), 200.0 µM (
), and
500 µM (
) of 8-BDB-TADP. Control mixtures contained
67.0 µM (
), 100.0 µM (
), 200.0
µM
), and 500.0 µM (
) of ADP in
place of 8-BDB-TADP. The experimental conditions are described in the
text. The percent of initial (zero time) enzyme activity is plotted on
a logarithmic scale along each ordinate, and time is
represented by the linear scale on each abscissa. This method
of plotting obscures the stimulation 3
-HSD activity by increasing
levels of 8-BDB-TADP (panelA). For that reason, the
initial 3
-HSD activity at each 8-BDB-TADP concentration is
provided here: 500 µM, 219 nmol/min/mg; 200
µM, 101 nmol/min/mg; 100 µM, 79 nmol/min/mg;
67 µM, 68 nmol/min/mg. Each plot is the result of
duplicate experiments.
The Kitz and Wilson analysis (9) determined an inhibition constant (K = 314 µM) and a first-order maximal rate
constant (k
= 7.8
10
s
) for the inactivation of isomerase by
8-BDB-TADP. In contrast to the first-order kinetics of isomerase
inactivation, the inactivation of 3
-HSD by 8-BDB-TADP exhibited an
intriguing reversal of the expected kinetics that could not be analyzed
by the Kitz and Wilson model.
The presence of 8-BDB-TADP in the
experimental incubation mixtures stimulated the 3-HSD activity at
zero time relative to the 3
-HSD activity in control mixtures that
contained ADP in place of 8-BDB-TADP (Fig. 1A). This
indirect effect followed a concentration dependence that was consistent
with the K
(338 µM) measured for
isomerase activation by 8-BDB-TADP. Because the inactivation data are
plotted as percent of zero time activity, the controls appear to
decrease as the ADP concentration increases. In fact, the control
3
-HSD activity was quite similar at all of the ADP concentrations
(mean ± S.D. = 67.1 ± 4.7 nmol/min/mg), and the
3
-HSD activity in the experimental mixtures increased as the
8-BDB-TADP concentration increased (values given in Fig. 1legend). In contrast, the isomerase controls are plotted
at 100% activity in Fig. 1B because aliquots of the
control and experimental mixtures are diluted 20-fold into the
isomerase assay mixture where saturating levels of isomerase substrate
steroid and activating nucleotide (NAD
) induce the
isomerase conformation. These assay conditions reverse the stimulation
of isomerase by 8-BDB-TADP in the enzyme inactivation mixture.
Stimulation by 8-BDB-TADP is evident in Fig. 1A because
the 3
-HSD assay conditions do not promote the isomerase
conformation to obscure the effect of 8-BDB-TADP on the enzyme protein.
Figure 2:
Protective effects of cofactors and
substrate steroids against the inactivation of 3-HSD and isomerase
by 8-BDB-TADP. 3
-HSD (panelA) and isomerase (panelB) activity were measured in incubations of
enzyme (1.0 µM) with 100.0 µM 8-BDB-TADP
alone (
) and in identical mixtures with 8-BDB-TADP plus 50.0
µM NADH (
), 150.0 µM NAD
(
), or substrate steroid (
, 10.0 µM pregnenolone for 3
-HSD or 150.0 µM
5-androstene-3,17-dione for isomerase). The unprotected control mixture
(
) contained ADP in place of 8-BDB-TADP, and the protected
control mixtures (
) also included the appropriate cofactor or
steroid. The percent of initial (zero time) enzyme activity is plotted
on a logarithmic scale along the ordinates, and time is
represented by the linear scale on each abscissa.
Figure 3:
Time-dependent activation of isomerase by
NADH. Purified 3-HSD/isomerase was preincubated (2.0 min) with
isomerase substrate (10.0 µM 5-pregnene-3,20-dione), and
NADH (2.4 µM) was added to start the reaction (
). In
an identical assay, the enzyme was preincubated in buffer, and the
reaction was started by adding a mixture of the substrate steroid and
NADH (
). In another identical assay, the enzyme was preincubated
with NADH, and the isomerase substrate was added to start the reaction
(
). Additional experimental conditions are described in the text.
The isomerase activity (nmol of progesterone formed) was measured at 15
equal intervals during the first minute and at 4 equal intervals during
the second minute to obtain the enzyme velocity versus time
plots. The half-time (t
) of the time-dependent
activation of isomerase was determined from the abscissa intercept of a straight line (not shown) extrapolated from the
linear portion of the plot (1-2 min). The values are the means
from duplicate experiments.
The spectrophotometric measurement of the activation of isomerase by 8-BDB-TADP did not reveal a time lag, suggesting that 8-BDB-TADP induces the conformational change in the enzyme more rapidly than NADH (data not shown).
Figure 4:
Stopped-flow fluorescence spectroscopy of
3-HSD/isomerase after the addition of NADH. Drive syringe 1
contained 1.2 µM pure enzyme in 0.02 M potassium
phosphate buffer, pH 7.4, 20% glycerol, 0.1 mM EDTA, 0.2%
Genapol C-100. Drive syringe 2 contained 20.0 µM NADH in
0.02 M potassium phosphate buffer, pH 7.4. After firing the
syringes at zero time, the reaction cell contained a 1:1 mixture of the
contents of each. Additional experimental details are described in the
text. The inset shows the first 10 s of the NADH-induced change in
fluorescence. The quenching of protein fluorescence was measured as
volts with positive values representing decreasing intrinsic
fluorescence. The figure shows a representative plot from three
determinations.
The
stoichiometry and dissociation constant (K) of the binding of NADH were
determined by measuring the stepwise decrease in the intrinsic
fluorescence of the enzyme (3.0 µM) during titration with
NADH (0.5-17.2 µM). From the Stinson and Holbrook
plot (17) in Fig. 5, the stoichiometry of 1.04 mol of
NADH bound/mol of enzyme dimer was calculated from
([NADH]/
intercept)/3.0 uM enzyme dimer. The K
of 4.9 µM NADH was
calculated from 1/slope of the best-fitting straight line plot.
Figure 5:
Determination of the stoichiometry and
dissociation constant for the binding of NADH to 3-HSD/isomerase
using fluorescence spectroscopy. The enzyme (3.0 µM) was
titrated with successive additions of NADH (0.5-17.2
µM). The decrease in intrinsic enzyme fluorescence
produced by titration with NADH was measured by excitation at 290 nm
and emission at 330 nm. Further experimental detail and the
construction of the plot are described in the text. The stoichiometry
was calculated from the abscissa intercept/3.0 µM enzyme, and the dissociation constant was calculated from 1/slope
of the best-fitting straight line through the points
(
).
It
was not possible to use 8-BDB-TADP in similar studies of stopped-flow
or intrinsic fluorescence spectroscopy because of the strong
ultraviolet absorbance of 8-BDB-TADP (extinction coefficient =
19,000 M cm
at
= 278 nm) and the high 8-BDB-TADP
concentrations (K
= 338 µM)
needed to perform the experiments.
As our studies of purified human placental
3-HSD/isomerase with affinity alkylators have
progressed(5, 7, 8, 18, 19, 20) ,
it has become increasingly clear that the two-step enzyme mechanism is
more complex than a dehydrogenase reaction followed by an isomerase
reaction along a single protein with separate substrate and coenzyme
sites for each activity. In addition to the NADH/NAD
protection(5, 8) and the secosteroid
inactivation (8) studies discussed above, NADH (20) and
pregnenolone (19) protected the same tryptic peptides (Arg-250,
Lys-175) in the enzyme from affinity radioalkylation by
2
-bromo[2`-
C]acetoxyprogesterone
(2
-[
C]BAP). Moreover, NADH shifted the
binding of 2
-[
C]BAP to radiolabel a
histidine in the Lys-135 peptide, while pregnenolone simply competed
with 2
-[
C]BAP for access to the Arg-250 and
Lys-175 peptides(19, 20) . This shift in affinity
radiolabeling that was produced by NADH protection, but not by
pregnenolone protection, provided further support for our hypothesis:
NADH formed by the 3
-HSD reaction induces a conformational change
in the enzyme protein that activates isomerase. The hypothesis has now
been definitively tested by measuring the inactivation of 3
-HSD
and isomerase with the NADH site-directed, affinity alkylating
nucleotide, 8-BDB-TADP, as well as by obtaining direct spectroscopic
measurements of the time-dependent activation of isomerase by NADH.
The mixed activator analysis and protection studies are consistent
with 8-BDB-TADP binding at the NADH site on the enzyme. NADH completely
protected against the inactivation of both 3-HSD and isomerase by
all other affinity alkylators we have studied (2
-BAP(5) ,
FSBA(7) , and secosteroid(8) ). Complete protection
against inactivation is an unusual observation and suggests that NADH
protected in these cases by inducing a conformational change in the
enzyme rather than by simple competition. The significant, but less
than complete, protection seen with 8-BDB-TADP plus the mixed activator
results suggest that NADH directly competes with this affinity
alkylator.
NAD failed to protect either activity
from 8-BDB-TADP. There has been no significant protection by
NAD
against any alkylator we have studied thus far,
with the notable exception of the inactivation of 3
-HSD by
FSBA(7) . NAD
slowed the FSBA inactivation of
3
-HSD by 3-fold but did not significantly protect isomerase from
inactivation by FSBA. Although both FSBA and 8-BDB-TADP are alkylating
analogs of adenosine, the respective alkylating groups are located at
``opposite'' ends of the adenosine molecule (8-position of
adenine in 8-BDB-TADP versus the 5`-position of ribose in
FSBA). FSBA is the weakest activator of isomerase in the group of
nucleotide-analogs studied (Table 1). Finally, FSBA inactivates
3
-HSD by the expected first-order kinetics (not
``reverse'' kinetics) because FSBA does not significantly
activate isomerase. The current study with 8-BDB-TADP indicates that
FSBA binds at the NAD
site when the enzyme is in the
3
-HSD conformation, whereas 8-BDB-TADP binds at the NADH site
after the 8-substituted nucleotide induces the enzyme to assume the
isomerase conformation.
Because 8-BDB-TADP is a highly efficacious
activator of isomerase, a definitive model has been developed to
explain the ``reverse'' kinetics of 3-HSD inactivation
by the affinity alkylating nucleotide. According to this model, the
concentration-dependent activation of isomerase by increasing levels of
8-BDB-TADP (I) converts progressively more molecules of enzyme
from the 3
-HSD conformation (E) into the isomerase form (E`
I). Enzyme alkylated in the isomerase form (E`
- I) retains significant dehydrogenase activity when an
aliquot from the alkylator/enzyme mixture is diluted 10-fold in the
3
-HSD assay cuvette, where the dehydrogenase conformation is
favored at pH 9.7 (3
-HSD optimum) with pregnenolone as substrate.
However, enzyme alkylated in the isomerase conformation has no activity
during the isomerase assay because the conformation is not shifted to
the dehydrogenase form under these incubation conditions (at the
isomerase optimal pH 7.5 with 5-pregnene-3,20-dione as substrate). This
model is illustrated by the following reaction scheme:
As the concentration of 8-BDB-TADP is increased, more alkylated
enzyme exists in the active E` - I form during the assay
used to measure 3-HSD inactivation. Thus, the induction of the
isomerase conformation (E`
I) by the reversible binding
of 8-BDB-TADP to the enzyme is directly responsible for the decrease in
the rate of 3
-HSD inactivation as the concentration of 8-BDB-TADP
increases.
At each of the 8-BDB-TADP concentrations used
(67-500 µM), a portion of the enzyme molecules
remains in the dehydrogenase form (E) during the inactivation.
Because formation of the reversible enzyme-alkylator complex induces
the isomerase conformation (E`I), the enzyme in the
dehydrogenase form is inactivated by 8-BDB-TADP via a bimolecular
mechanism (E + I
E - I).
Based on the measurements of 3
-HSD inactivation, enzyme alkylated
by 8-BDB-TADP while in the dehydrogenase conformation (E - I) has no activity in the 3
-HSD assay and
partial activity in the isomerase assay.
The inactivation of isomerase by 8-BDB-TADP fits the equation for biphasic enzyme inactivation(10) ,
where kfast and kslow represent the values of k = 0.693/t
that
were measured for the inactivation of isomerase and 3
-HSD,
respectively, at each concentration of 8-BDB-TADP (determined from Fig. 1, A and B). The variable F represents the fractional residual activity of isomerase. The
calculated curves fit the data points measured for the inactivation of
isomerase (Fig. 6B) until less than 20% of the initial
activity remains. At this point, the observed data points are higher
than the predicted curves because all available enzyme in the isomerase
form (E`
I) has been inactivated as E` - I, leaving a mixture of E and E - I.
Because the biphasic inactivation equation assumes that the alkylated
enzyme (E` - I or E - I) has no activity, the observed data points exceed the values
of the calculated points due to the partial isomerase activity of
enzyme alkylated in the 3
-HSD form (E - I).
Figure 6:
The inactivation of 3-HSD and
isomerase by 8-BDB-TADP fit curves predicted by equations for one-phase
and two-phase enzyme inactivation, respectively. The 3
-HSD (panelA) or isomerase (panelB)
activity of the enzyme (1.0 µM) was inactivated by 67.0
µM (
), 100.0 µM (
), 200.0
µM (
), and 500 µM (
) of
8-BDB-TADP. The inactivation of 3
-HSD fits the equation for
single-phase enzyme inactivation, E/E
= (1 - P)e
+ P, where kobs represents the value
of k
= 0.693/t
that was measured for 3
-HSD inactivation over time (t) at each 8-BDB-TADP concentration (determined from Fig. 1A). The inactivation of isomerase fits the
equation for biphasic enzyme inactivation, E/E
= (1 - F)e
+ (F)e
,
where kfast and kslow represent the values of k
= 0.693/t
that
were measured for the inactivation of isomerase and 3
-HSD,
respectively, at each concentration of 8-BDB-TADP (determined from Fig. 1, A and B). The remaining parameters and
the significance of the fits are discussed in the text. Fractional
enzyme activity (E/E
, E
= 1.0) is plotted on a logarithmic scale along each ordinate, and time is represented by the linear scale on each abscissa.
The equation for biphasic enzyme inactivation
overestimates the observed rate of 3-HSD inactivation. However,
the data fits the equation for single-phase enzyme inactivation (Fig. 6A),
where kobs represents the value of k = 0.693/t
that was measured for
3
-HSD inactivation at each 8-BDB-TADP concentration (determined
from Fig. 1A). The variable P represents the
fractional residual 3
-HSD activity when an inactivation plateau is
reached. Because 8-BDB-TADP decomposes relatively slowly (t
= 56 min) compared to the rates of
inactivation measured for 3
-HSD, reagent decomposition causes the
observed data points to exceed the predicted values only at the lower
8-BDB-TADP concentrations (67 and 100 µM) after 20 min of
inactivation.
The need to switch from a two-phase to single-phase
equation to fit the data obtained for the inactivation of 3-HSD
supports the concept that enzyme alkylated in the isomerase
conformation has full activity in the 3
-HSD assay. Because a
greater proportion of enzyme molecules are in the isomerase
conformation as the 8-BDB-TADP concentration increases (Table 1),
higher concentrations of 8-BDB-TADP inactivate 3
-HSD more slowly
than lower concentrations to yield the ``reverse'' kinetics
of 3
-HSD inactivation.
Our hypothesis that NADH activates
isomerase by inducing a conformational change in the enzyme protein is
indirectly supported by this affinity labeling study: 8-BDB-TADP binds
at the NADH site, activates isomerase, and produces the reverse
kinetics of 3-HSD inactivation. Moreover, direct evidence for the
NADH-induced conformational change has been obtained by observing the
time dependence of both the activation of isomerase (Fig. 3) and
the quenching of intrinsic protein fluorescence by NADH (Fig. 4). The fact that the time frame for the fluorescence
change (t
= 85 s) is longer than for the
activation of isomerase by NADH (t
= 20
s) suggests that a point is reached during the conformational change
where the isomerase substrate is brought into proper juxtaposition with
the amino acid residues that catalyze the reaction. Once that threshold
is reached, isomerization proceeds at the maximal rate.
The stoichiometry of 1 mol of NADH bound/mol of enzyme dimer can be interpreted in two ways: 1) NADH induces the isomerase conformation in just one of the two subunits or 2) both subunits form a single NADH site when the enzyme is in the isomerase form. Whether one or both subunits participate in the isomerase activity will require studies of tertiary and quaternary structure by nuclear magnetic resonance spectroscopy or x-ray crystallography.
The inactivation data
obtained with the NADH site-directed affinity alkylator, 8-BDB-TADP,
complement the direct measurements of the NADH-induced activation of
isomerase to validate our proposed mechanism for the sequential
3-HSD/isomerase activity. As the 3
-HSD activity oxidizes the
3
-hydroxy-5-ene steroid (pregnenolone or dehydroepiandrosterone)
to the 3-oxo-5-ene intermediate, NAD
is reduced to
form NADH. This NADH induces a conformational change in the enzyme
protein that activates isomerase to produce the 3-oxo-4-ene steroid
(progesterone or androstenedione). After the product steroid and NADH
dissociate, the enzyme converts back to the dehydrogenase form and can
again catalyze the reaction sequence. Understanding how the enzyme
shifts from the first to the second reaction in the sequence will help
us evaluate the relationship between the individual reaction mechanisms
for 3
-HSD and isomerase, which are currently being studied in our
laboratory.