(Received for publication, July 13, 1995; and in revised form, October 20, 1995)
From the
Human type II inosine 5`-monophosphate dehydrogenase has been
purified to homogeneity from an Escherichia coli strain that
express large quantities of the enzyme from the cloned gene. Steady
state kinetic studies have been used to characterize the activation by
monovalent cations, including Li, Na
,
K
, Rb
, Cs
,
Tl
, NH
, and
N(CH
)
. The enzyme has less
than 1% of the maximal activity in the absence of an added monovalent
cation, such as K
, Na
,
Rb
, Tl
, or
NH
. The enzyme is activated by
K
and Tl
at lower concentrations than
those of other monovalent cations. Li
and
N(CH
)
do not activate the
enzyme, nor do they inhibit the K
-activated enzyme,
implying that ionic radius is important in binding selectivity. The K
values for both substrates and V
differ with different monovalent cations.
Initial velocity and product inhibition kinetic data are consistent
with an ordered steady state mechanism in which the enzyme binds
K
first, IMP second, and then NAD; the product NADH is
released before xanthosine 5`-monophosphate. Substrate and product
binding experiments support this mechanism and show the presence of one
substrate binding site per subunit. Several rate constants were
obtained from a computer simulation of the complete steady state rate
equation.
Inosine 5`-monophosphate dehydrogenase (IMPDH; ()IMP-NAD oxidoreductase, EC 1.2.1.14) catalyzes the
reaction:
which is the rate-limiting step in the biosynthesis of guanine nucleotides(1, 2) . IMPDH activity has been linked to malignancy because enzyme levels are greatly elevated in tumor tissues and the levels are correlated with cell growth rates(3, 4, 5) . There are two isozymes of human IMPDH, denoted types I and II (or IMPDH-h1 and IMPDH-h2)(6) . IMPDH-h2 expression is selectively up-regulated in leukemias and brain tumor tissues(7, 8) . Based on these observations, IMPDH has been recognized as a target for antitumor agents(9) .
Isoforms of IMPDH have been purified from a
variety of eukaryotic and prokaryotic sources; all are tetrameric with
subunit molecular weights of 56
kDa(10, 11, 12, 13, 14) .
Both IMPDH-h1 and IMPDH-h2 subunits have 514 amino acids, and they have
84% sequence identity(6) . IMPDH-h2 is inactivated by
6-chloro-purine ribotide, which reacts with Cys-331 in the IMP binding
site, providing the only evidence for the location of the IMP binding
site(6, 15, 16) . A monovalent cation, such
as K
, Na
, Rb
,
NH
, or Tl
, is required
for maximal activity, but the mechanism and extent of the activation
are unknown. Monovalent cations of different radii show different
abilities to activate the enzymes from Bacillus subtilis and
sarcoma 180 cells(17, 18) . An absolute requirement
for a monovalent cation activator has not yet been demonstrated,
perhaps due to the use of sodium or potassium salts of substrates or
other components in assay solutions, and thus 10% or more of the
maximum activities have been reported in experiments in which no
monovalent cations were intentionally added(17, 18) .
A steady state ordered sequential Bi Bi mechanism in which IMP binds
before NAD and XMP is released after NADH is commonly used to describe
the IMPDH-catalyzed reaction(19) . Because this mechanism does
not include the monovalent cation, it is not complete. Another
partially random rapid equilibrium mechanism including
K, IMP, and NAD was proposed by Morrison and
co-workers (20) for the enzyme from Aerobacter
aerogenes. In this mechanism, K
and IMP are
proposed to bind randomly to the enzyme, whereas NAD does not bind
unless K
or both K
and IMP are bound.
In this study, we constructed an Escherichia coli expression system for IMPDH-h2 and purified the recombinant
protein to homogeneity. The activation of the recombinant IMPDH-h2 by
various monovalent cations has been characterized and a larger than
100-fold activation by a monovalent cation, such as K,
was found. A steady state kinetic mechanism including the monovalent
cation activation of IMPDH-h2 is proposed based on initial velocity and
product inhibition studies. Substrate and product equilibrium binding
experiments were performed to directly determine binding affinities and
to provide further evidence supporting the proposed mechanism.
NAD, NADH, alkaline phosphatase, and the free acid of IMP
were purchased from Sigma. The sodium salt of XMP (Sigma) was converted
into an XMPTris
salt by passage through a Dowex
50
Tris
cation exchange column. Elemental
analyses were performed by the inductively coupled argon plasma method
at the chemical analysis laboratory of the University of Georgia. The
elements analyzed included potassium, sodium, zinc, copper, manganese,
iron, magnesium, and phosphorus. All pH values, except where otherwise
specified, were measured at room temperature.
The 1.5-kilobase pair fragment obtained from the
polymerase chain reaction was isolated from 1% agarose gel (21) and digested with NdeI and BglII
restriction enzymes according to the manufacturer's
recommendations (BioLabs). The resulting fragment was ligated into the NdeI-BamHI sites of the pET12B vector (from
Novagen), transformed into the strain MV1190, and plated on
LB-Ampicillin media with
isopropyl-1-thio--D-galactopyranoside and X-Gal added.
Plasmids from transformants were mapped by restriction digestion, and
the complete DNA sequence was determined using an Applied Biosystems
automated DNA sequencer. A clone called pIMPDH-h2 had the expected
restriction map and DNA sequence and was transformed into strain
BL21(DE3) for further study.
The specific activity of the purified
IMPDH-h2 determined by the standard assay was 0.68 µmol (NADH
formed)/mg/min. The concentration of the purified IMPDH-h2 was
determined by UV absorption; the absorbance at 280 nm for a 1 mg/ml
IMPDH-h2 concentration was found to be 0.465 using the 205 nm/280 nm
ratio method of Scopes(23) . An overall yield of 150 mg
IMPDH-h2 from 10 g of cells was obtained.
The concentration of the unbound NADH was measured from its absorbance at 340 nm. However the concentrations of unbound XMP, IMP, and NAD could not be directly determined from their UV absorption due to interference from the UV absorption of the dithiothreitol in the buffer, which slowly oxidized to form variable amounts of a UV-absorbing cyclic disulfide. To measure the concentration of unbound XMP, the phosphate group on XMP was enzymatically removed by alkaline phosphatase, and the concentration of the released phosphate was determined colorimetrically(25) .
The concentrations of the unbound IMP and NAD were determined by the
method described below. The equilibrium of the IMPDH-catalyzed reaction
lies far toward products(18) . In an assay solution when the
concentration of one substrate was more than 10 times the concentration
of the second substrate, the NADH concentration produced by the
reaction was found to be equal to the starting concentration of the
second substrate. The unbound IMP or NAD from the Centricon device was
put in a cuvette, and the other substrate was added at a concentration
known to be more than 10 times that of the unbound substrate.
K was also added to a concentration of
10
mM. The reaction was started by the addition of IMPDH-h2. The
final NADH concentration was determined from the increase in absorbance
at 340 nm. The lack of a need of a reference sample is an advantage of
this method.
Binding experiments were done with five different
concentrations of substrate or product. Total enzyme concentrations (E) and total substrate or product concentrations (L
) were known, and unbound substrate or product
concentrations (L) were measured. The data were fit to the
equation: E
/(L
- L) = (K
/N)(1/L)
- 1/N to determine the number (N) of binding
sites per IMPDH-h2 molecule and the dissociation constant (K
) of the substrate- or product-IMPDH-h2
complex(26) .
The activity of
IMPDH-h2 was determined at a series of different pH values to find the
optimal pH as shown in Fig. 1. The pH value of the assay buffer
that provides maximal IMPDH-h2 activity is 7.7 at 37 °C, which for
this buffer corresponds to pH 8.1 at room temperature. In all other
experiments, assay buffers were adjusted to pH 8.1 at room temperature.
A computer fit of the pH rate profile gave apparent pK and pK
values of 6.8 and 8.5 (Fig. 1).
Figure 1:
The
dependence of IMPDH-h2 activity on pH. Assay solutions contained 100
mM Tris-HCl, 0.2 mM IMP, 0.2 mM NAD, 10
mM K, and 32 nM IMPDH-h2. The data
are fit to the equation: IMPDH-h2 activity (V) = C/(1 +
[H
]/K
+ K
/[H
]), where C, K
, and K
are 5.2 µM/min, 1.5
10
M, and 3.0
10
M,
respectively.
The activation of IMPDH-h2 by
various monovalent cations was characterized. Fig. 2shows the
relation between IMPDH-h2 activity and monovalent cation concentration
for K, Na
, Rb
,
NH
, Tl
, and
Cs
. For each monovalent cation, the activity of
IMPDH-h2 increases with increasing cation concentration and reaches an
optimum; higher cation concentrations reduce the activity. K
produces the highest activity, whereas Tl
produces optimal activity at the lowest concentration. The
optimal concentrations for K
and Tl
,
which have similar ionic radii, are
20 mM and
5
mM, respectively. Other cations have optimal concentrations of
100 mM. Li
and
N(CH
)
were found not to
activate the enzyme, and concentrations up to 100 mM did not
inhibit the enzyme activated by 10 mM K
.
These results suggest that the affinity of IMPDH-h2 for the monovalent
cation is sensitive to the size of the ion.
Figure 2:
IMPDH-h2 activities measured at various
concentrations of monovalent cations K,
Na
, Rb
,
NH
, Tl
, or
Cs
. Solutions were buffered with 100 mM Tris-HCl except for experiments with Tl
, where
100 mM Tris-acetic acid was used. In all cases 0.2 mM IMP, 0.2 mM NAD, and 32 nM IMPDH-h2 were
present. The cations were added as chloride salts except for the use of
thallium acetate.
The dependence of
IMPDH-h2 activity on concentrations of IMP and NAD were studied in the
presence of Na, K
,
Tl
, Rb
, and NH
at their optimal concentrations. The data were fit to the
equation for the steady state ordered sequential Bi Bi mechanism in
which IMP binds to the enzyme before NAD. The kinetic constants for
IMPDH-h2 activated by the five monovalent cations are shown in Table 1. The kinetic constants for the
K
-activated IMPDH-h2 are comparable with those
recently reported by Carr et al.(19) using enzyme
obtained from a different expression system and purification
scheme(19) . There appears to be no simple relation between the
substrate K
values, V
, and
ionic radius, although very small (Li
) or very large
(N(CH
)
) cations apparently do
not bind.
Fig. 3shows double reciprocal plots of the
dependence of IMPDH-h2 activity on the concentrations of
K, IMP, and NAD. Each plot consists of nonparallel
straight lines intersecting at a point off either axis. Fig. 4shows the double reciprocal plots for product inhibition
experiments. XMP shows competitive inhibition with respect to IMP and
noncompetitive inhibition with respect to K
and NAD.
NADH inhibits noncompetitively with respect to K
, IMP,
and NAD. No cation that inhibits by competing with K
has been found. These data are consistent with an ordered
sequential kinetic mechanism.
Figure 3:
Double reciprocal plots of the dependence
of IMPDH-h2 activity on the concentrations of K, IMP,
and NAD. Conditions other than those indicated were the same as those
in the standard assay.
Figure 4:
Double reciprocal plots of the dependence
of IMPDH-h2 activity on the concentrations of K, IMP,
NAD, NADH, and XMP. Conditions other than those indicated were the same
as those in the standard assay.
Figure 5:
IMP and NADH binding to IMPDH-h2 at 37 or
4 °C. The data were fit to the equation E/(L
- L)
= (K
/4)(1/L) -
¼, for the model of four IMP or NADH binding sites per IMPDH-h2
tetramer.
A kinetic mechanism including the monovalent cation activator
has not previously been elucidated for human IMPDH(19) . The
kinetic data shown in Fig. 3and Fig. 4are not
consistent with the partially random rapid equilibrium reaction
mechanism suggested by Morrison and co-workers (20) for IMPDH
from A. aerogenes because the common intersection (Fig. 3E) in plots of 1/V versus 1/[NAD] at different concentrations of K is not on the vertical axis. The competitive inhibition by XMP
with respect to IMP (Fig. 4C) and the characteristic
off-axis common intersections in Fig. 3(D and F) and 4 (D, E, and F) are the
patterns predicted by the steady state ordered sequential Bi Bi
mechanism in which IMP binds first and XMP is released
last(20) . This part of the mechanism has been proposed by
other
workers(13, 19, 28, 29, 30, 31, 32) .
A more complete mechanism must take into account the monovalent
cation, which this study shows to be an essential activator for
IMPDH-h2. Based on the dependence of IMPDH-h2 activity on K and substrate concentrations shown in Fig. 3and product
inhibition results shown in Fig. 4, a steady state reaction
mechanism including the activation of IMPDH-h2 by a monovalent cation
is proposed in Fig. 6. In the productive sequence, IMPDH-h2
binds the monovalent cation first and then binds IMP, and NAD is bound
last. NADH is released before XMP. Because no inhibitors that compete
with K
are known, it is not clear whether the
monovalent cation activator is required to dissociate in each catalytic
cycle. Kinetic studies indicate that the free IMPDH may also bind IMP
and XMP to form dead end complexes, and the formation of binary
enzyme
IMP and enzyme
XMP complexes has been verified by
equilibrium binding studies. If no products are present, the dependence
of IMPDH activity (V) on the monovalent cation (M),
IMP (A), and NAD concentration (B) is described by . In the presence of XMP or NADH inhibitors, the relation
is described by or , where Q and P are concentrations of XMP and NADH, respectively. The
constants C with different subscripts (see
``Appendix'') are combinations of rate constants and total
enzyme concentration (E
). These equations were
derived using the King-Altman method(33) .
Figure 6:
Steady state mechanism proposed for the
IMPDH-catalyzed reaction. E, M, A, B, P, and Q represent IMPDH monovalent
cation such as K, IMP, NAD, NADH, and XMP,
respectively.
predicts an off-axis common intersection for each double reciprocal plot as found in the results shown in Fig. 3. predicts the competitive inhibition of XMP with respect to IMP as shown by the common intersection on the vertical axis in Fig. 4C. The off-axis common intersections in Fig. 4(A, B, D, E, and F) are all consistent with equations 2 and 3. Thus the initial velocity data (Fig. 3) and product inhibition data (Fig. 4) agree with the proposed mechanism shown in Fig. 6. The double reciprocal plots of initial velocity and product inhibition obtained by Anderson and Sartorelli for IMPDH from sarcoma 180 ascites tumor cells are also consistent with the mechanism proposed here(18) .
Evidence supporting the mechanism in Fig. 6was provided by equilibrium binding experiments. The EA
complex (IMPIMPDH-h2), and the EQ complex (XMP
IMPDH-h2)
were shown to form, and the data were consistent with four IMP binding
sites per IMPDH-h2 tetramer. This agrees with results obtained by Wu
and co-workers from IMPDH inactivation by 6-Cl-IMP, which suggested one
IMP binding site per IMPDH subunit(16) . The 2-fold greater
affinity for IMP found in the presence of 10 mM K
is consistent with the K
value, which
reflects the affinity of IMP for the binary enzyme
K
complex (Table 1). It is as yet not clear whether there is
a direct interaction between K
and IMP or whether
K
mediates a conformational change of IMPDH-h2. NAD
and NADH did not bind detectably at 37 °C. Although NADH binding
was detected at 4 °C, this E
NADH complex is apparently not on
the primary kinetic pathway.
The individual rate constants and
dissociation constants determined by the computer simulation fitting
the steady state rate equation to 150 rate measurements at varying
concentrations of K, IMP, NAD, XMP, and NADH as
described under ``Experimental Procedures'' are shown in Table 3. Other rate constants from the simulation have large
standard deviations and are not shown. K
and K
are very close to the dissociation constants of
XMP and IMP determined by direct binding experiments (Table 2).
This indicates that the results (Table 3) from the computer
simulation are reasonable and will provide a framework for future
studies.
An essential role of a monovalent cation such as
K, Na
, Rb
,
Tl
, and NH
for IMPDH-h2
activity is indicated by this study. Removal of the monovalent cation
from IMPDH-h2 by dialysis does not cause an irreversible loss of
activity of the enzyme and does not destroy its tetrameric structure.
The binding and release of the monovalent cation is thus a reversible
process. K
and Tl
are better IMPDH-h2
activators than other monovalent cations in the sense that they produce
maximal activity at about 10 times lower concentrations than other
monovalent cations, as shown in Fig. 2. This suggests a size
selectivity for ion binding. The maximal activities achieved with the
tested cations are all of the same order of magnitude. Because the
rate-limiting step of the reaction is not yet known, it is possible
that different cations have different effects on intrinsic steps that
are partially masked by cation-insensitive steps. Recent
crystallographic studies demonstrated conformational changes in
diakylglycine decarboxylase when it bound different monovalent cations
including K
, Rb
, Na
,
and Li
, and the conformational change was shown to be
critical for the activation by K
and Rb
and inhibition by Na
and Li
(34, 35) . The variation of kinetic constants in
the presence of different monovalent cations (Table 1) observed
in this study may suggest that different IMPDH-h2 conformational
changes are caused by the binding of ions of different ionic radii. The
dependence of kinetic constants on ionic radii of the monovalent
cations as shown in Table 1may imply that the ionic radius is
critical in determining the monovalent cation binding affinity and the
nature of the putative conformational change caused by the monovalent
cation binding.
The complete steady state rate equation for the mechanism
shown in Fig. 6: V = (kk
k
k
AB - k
k
k
k
PQ)E
K
K
M/[(k
+ k
)K
k
k
K
K
+ (k
+ k
)k
k
K
K
M + (k
+ k
)K
k
k
K
A + K
k
k
k
K
K
B + (k
+ k
)k
k
K
K
MA + k
k
k
K
K
MB + K
k
k
k
K
AB + (k
+ k
)k
k
K
K
MAB + K
k
k
k
K
K
P + (k
+ k
)K
k
k
K
Q + k
k
k
K
K
MP + K
k
k
k
K
AP + (k
+ k
)k
k
K
K
MQ + K
k
k
k
K
BQ + k
k
k
K
K
MAP + k
k
k
K
K
MBQ + k
k
k
K
K
MABP + K
k
k
k
K
PQ + (k
+ k
)k
k
K
K
MPQ + k
k
k
K
K
MBPQ].
The coefficients in , , and are: C
, (k
+ k
)/E
k
k
; C
,
1/E
k
; C
, K
/E
k
; C
, K
(k
+ k
)/E
k
k
; C
,
1/E
k
K
; C
, K
(k
+ k
)/E
k
k
K
; C
,
1/E
K
k
; C
, K
K
K
/E
K
k
K
; C
, K
K
K
/E
K
k
; C
, k
/E
k
k
K
; C
, (k
+ k
)/E
k
k
; C
, K
K
(k
+ k
/ E
k
k
K
; C
,
K
K
k
+ k
)/E
k
k
; C
, K
/E
k
K
; C
, K
K
(k
+ k
)/E
k
k
K
; C
, K
/E
K
k
; C
, K
K
/E
K
k
. Ks and ks are dissociation constants and individual
rate constants as shown in Fig. 6.