From the Department of Human Biological Chemistry and Genetics,
University of Texas Medical Branch, Galveston, Texas 77555-1055
A fundamental issue in allosteric regulatory
enzymes is the identification of pathways of signal transmission.
Rabbit muscle and kidney pyruvate kinase isozymes are ideal to address
this issue because these isozymes exhibit different enzymatic
regulatory patterns, and the sequence differences between these
isozymes have identified the amino acid residues that alter their
kinetic behavior. In an earlier study, Cheng et al. (Cheng,
X., Friesen, R. H. E., and Lee, J. C. (1996)
J. Biol. Chem. 271, 6313-6321), reported the effects
of a threonine to methionine mutation at residue 340 in the muscle
isozyme. In this study, the same mutation was effected in the kidney
isozyme. Qualitatively, the same negative effects are observed in both
isozymes, namely a significant decrease in catalytic efficiency and
decrease in apparent affinity for phosphoenolpyruvate but no change in
affinity for ADP, and a decrease in responsiveness to the presence of
effectors, be it activator or inhibitor. Because the diversity in the
primary sequence between these two isozymes does not alter the negative
impact of the T340M mutation, it can be concluded that this mutation
exerts a dominant, negative effect. The negative effects of T340M
mutation on the kinetic properties imply that there is communication
between residue 340 and the active site. Residue 340 is located at the
1,4 subunit interface; however, a T340M mutation enhances the
dimerization affinity along the 1,2 subunit interface. Thus, this study
has identified a communication network among the active site, residue 340, and the 1,2 subunit interface.
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INTRODUCTION |
Mammalian pyruvate kinase (ATP:pyruvate
2-O-phosphotransferase, EC 2.7.1.40)
(PK)1 is a key regulatory
glycolytic enzyme which catalyzes the transphosphorylation from
phosphoenolpyruvate (PEP) to ADP. One of the enzymatic products, pyruvate, is situated at a major metabolic junction between the metabolic pathways of carbohydrates, amino acids, and lipids. Thus, a
tight regulatory control of PK is critical to proper cellular function.
PK activity in mammalian cells is regulated by two different
mechanisms. The first mechanism occurs at the level of expression. The mammalian genome contains two distinct genes coding for four different enzymes with PK activity. The M1- and
M2-type isozymes of PK are produced from one gene by
alternative RNA splicing (1). The R- and L-type PK isozymes are
products of a different gene with two different promoters. The R- and
L-type mRNAs differ only in their 5'-terminal sequences (2). These
four isozymic forms of PK are expressed in a tissue-specific manner
(3). The M1-type PK is the major isozyme of cardiac muscle
and brain and is the only isozyme found in adult skeletal muscle. The
M2-type PK is widely distributed throughout the body and is
the major isozyme derived from kidney and leukocytes. The L-type PK is
the major isozyme in the liver, and the R-type isozyme is isolated from erythrocytes.
The second mechanism of control of PK activity is through allosteric
regulation. PK isozymes are regulated by a host of allosteric inhibitors and activators (3). The M2-, R-, and L-type
isozymes are homotropically activated by PEP and heterotropically
activated by fructose-1,6-bisphosphate (FBP). The M1-type
isozyme is activated by PEP and FBP only in the presence of the
inhibitor L-phenylalanine (Phe), which is an inhibitor of
all isozymes. In addition, the catalytic activity of the L-type isozyme
is known to be allosterically regulated by phosphorylation (4). The
four PK isozymes have distinct kinetic characteristics. Thus, these
isozymes are genetic products to accommodate the various metabolic
requirements demanded by the different tissues.
In general, the current knowledge of the molecular mechanisms of
allosteric regulation at the atomic level is lagging far behind that of
the molecular mechanism of catalysis. Whereas a catalytic site usually
comprises a part of the enzyme, allosteric control is transmitted
over a long range, thus significantly increasing the number of possible
residues involved in regulation. It is, therefore, difficult to
identify the structural elements that play an important role in the
regulation of PK and the propagation of these allosteric signals.
Recently, an approach was successfully applied to increase the
efficiency of identifying structural elements involved in the
regulation of PK (5). The approach is based on the assumption that the
molecular mechanism of allosteric regulation is conserved for all
mammalian PK isozymes. This hypothesis is based on the structural
homology among mammalian PK isozymes and their qualitatively similar
functional behavior. It follows from this hypothesis that structural
elements involved in the regulation of PK have similar functional
significance in the other mammalian PK isozymes. Guided by this
hypothesis, genetic information from different PK isozymes is recruited
to identify the targets for mutational studies. Thus far, 55 different
mutations have been described for the human R-type PK isozyme from
patients with PK-deficient hemolytic anemia (6). Mutant PK
Tokyo/Nagasaki/Beirut was identified by genetic studies of erythrocyte
PK in hereditary nonspherocytic hemolytic anemia patients (7-9). This
mutant involves a threonine to methionine at a residue equivalent to
340 in rabbit kidney PK (RKPK). Threonine 340 or its equivalent is
conserved in all known aligned PK sequences (10). Based on the crystal
structure of RMPK, it is located outside of the active site (11). Fig. 1 shows a schematic representation of the
location of T340M mutation relative to the active site and the subunit
interfaces. The characterization of the T340M mutation in rRMPK has
been reported (5). Characterization of the same mutant in the rRKPK
will not only provide information to address the validity of the
hypothesis of an evolutionary conserved allosteric mechanism, it will
also provide information on the dominance of this mutation in the
presence of a background of sequence differences between rRKPK and
rRMPK. The ability of rRKPK to undergo subunit assembly provides a
unique opportunity to monitor the changes in energetics at the subunit
interface as a result of the T340M
mutation.2 The T340M rRKPK
mutant is characterized by kinetic and physical studies. The results
are analyzed to define the sites that communicate with each other, and
a structural model for the transmission of the allosteric signal is
discussed.

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Fig. 1.
Schematic diagram of the T340M rRKPK tetramer
looking down two 2-fold axes, constituting the 1,4 intersubunit
interface (A-AXIS) and the 1,2 subunit interface
(B-AXIS), respectively. The T340M mutation site in
each subunit is marked. The two pairs of dimer are in different shades
of gray. The sequence that is different between RMPK and
RKPK is in black along the B axis. The approximate distance
between the T340M mutation and the pyruvate molecule in the active site
is 24 Å, between the T340M mutation and K421 (1,2 subunit interface)
is 39 Å, and between K421 and the active site is 45 Å. These
distances are based on the x-ray crystallographic structure of RMPK
(11).
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EXPERIMENTAL PROCEDURES |
Materials--
Lactate dehydrogenase, disodium salt of ADP,
phosphoenolpyruvate, Tris base, and Tris-HCl were purchased from
Boehringer Mannheim. Reduced nicotinamide adenine dinucleotide (NADH),
L-phenylalanine (Phe), potassium chloride, sodium chloride,
and phenylmethylsulfonyl fluoride were all obtained from Sigma
Biochemicals. Mono- and dibasic potassium phosphate were purchased from
Fisher. [35S]dATP was purchased from Amersham Pharmacia
Biotech. Oligonucleotides were purchased from Genosys Biotechnologies,
Inc.
Site-directed Mutagenesis--
The mutagenic oligonucleotide
used for constructing the specific point mutant is as follows.
The underlined sequence is the mutagenic nucleotide with the
corresponding wild type sequence labeled below.
Mutagenesis of the PK gene was performed directly with the
double-stranded plasmid pRK-PK (13)2 using the published
procedure of Cheng et al. (5). Briefly, two oligonucleotide
primers, a mutagenic primer and a selection primer, were simultaneously
annealed to one strand of the denatured pRK-PK. The mutagenic primer,
containing the sequence shown above, was designed to introduce the
desired site-specific mutation into the rRKPK gene, whereas the
selection primer with the nucleotide sequence of 5'-TTTCA CACCG CAGCT
GGTGC ACTCT C-3' was to mutate a unique NdeI restriction
site in the pKK233-3 vector. The complementary strand, which
incorporated both primers, was then synthesized by T4-DNA polymerase
and circularized by T4-DNA ligase. Plasmids, propagated in a
repair-deficient Escherichia coli strain BMH 71-18 mutS,
were isolated. Screening for mutant plasmids was conducted by digestion
with NdeI. Wild type plasmids, containing an intact NdeI site, were linearized whereas mutant plasmids, lacking
the NdeI site, were resistant to the digestion. The reaction
mixture was transformed into JM 105 cells. Plasmids were isolated from culture of individual transformants and screened by NdeI
digestion. More than 50% of the plasmids that lacked the
NdeI site also contained the desired point mutation, as
confirmed by DNA sequencing of these plasmids.
DNA sequencing of double-stranded DNA was performed by the
dideoxynucleotide chain termination method (14) using Sequenase version
2.0 (U. S. Biochemical Corp.).
Overexpression of RKPK E. coli--
JM105 cells, containing the
plasmid pRK-PK, with the T340M mutation were grown overnight in M9
minimal medium containing 100 µg/ml ampicillin and were inoculated
1/100 (v/v) into NZCYM medium containing 100 µg/ml ampicillin.
Culture was grown at 37 °C until the optical density at 600 nm
reached 0.7-1.0, at which point expression was induced by the addition
of isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 120 µg/ml. After 12 h of induction, the cells
were harvested by centrifugation in a Sorvall RC-5C centrifuge using
the GS-3 rotor at 6000 rpm for 20 min.
Protein Purification and Preparation--
All purification steps
were performed at 4 °C except the SP-Sepharose chromatography, which
was performed at room temperature using the published procedure of
Cheng et al. (5) with the following minor modifications: 1)
the final concentration of polyethyleneimine was 0.35%, and 2) all
buffer solutions contained 1 mM DTT and 0.4 mM
FBP, the addition of which stabilizes the enzyme and ensures reproducible chromatographic behavior. Oxidation and the absence of FBP
affect the oligomeric state of the enzyme, resulting in longer
retention times on both chromatographic columns. Buffer A was 20 mM Tris-base, 100 mM KCl, 15% (v/v) glycerol,
1 mM DTT, 0.4 mM FBP, 0.1 mM EDTA,
and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.9. Buffer B
was 5 mM KPi, 1 mM EDTA, 1 mM DTT, 0.4 mM FBP, 5 mM
MgSO4, 100 mM KCl, pH 7.5. Buffer C was the
same as buffer B with no KCl and at pH 6.0.
Recombinant RKPK was desalted before all experiments by filtration
using a Superdex-200 AG on a FPLC system. The ammonium sulfate
precipitate was concentrated by centrifugation at 14,000 rpm in an
Eppendorf tabletop centrifuge for 5 min, and the pellet was resuspended
in up to 300 µl of the appropriate buffer before loading onto the
filtration column. Protein concentration was determined by absorbance
at 280 nm, using an absorptivity of 0.54 ml/(mg·cm) (15).
Enzyme Kinetics--
The enzymatic activity of T340M rRKPK was
determined by the lactate dehydrogenase-coupled enzyme assay (16). The
reaction occurs in a solution consisting of 50 mM Tris
base, 72 mM KCl, 7.2 mM MgSO4, 0.3 mM NADH, 10 µg/ml lactate dehydrogenase, and varying
amounts of Phe and FBP as indicated. The final concentration of ADP or
PEP in the assay mixture was fixed at 2 or 20 mM,
respectively, while varying the concentration of the other substrate.
After adjusting the pH of the assay mix to 7.5 at 23 °C, lactate
dehydrogenase that was equilibrated with TKM buffer (50 mM
Tris, 72 mM KCl, 7.2 mM MgSO4, pH
7.5) was added and the assay mix was finally brought to the desired
volume. The reaction was started by the addition of 2-4 µl of PK to
1 ml of assay solution that had been equilibrated at 23 °C. The
amount of PK present in the assay was about 0.5 µg. The decrease in
absorption at 340 nm was followed as a function of time with a Hitachi
U-2000 spectrophotometer to obtain
, the observed steady-state
kinetic velocity. All data sets were fitted to the modified version of
Hill equation as shown in Equation 1 (17).
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(Eq. 1)
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Vmax is the maximal velocity of each data
set, [S] is the concentration of the variable substrate, n
is the Hill coefficient, and Kapp is a complex
steady-state kinetic equilibrium constant that is equivalent to the
Km in the Michaelis-Menten equation, where
n = 1.
SDS-Gel Electrophoresis--
SDS-10% polyacrylamide gel
electrophoresis was performed according to the method of Laemmli (18),
followed by staining with Coomassie Blue. The markers used for
molecular mass determination were: phosphorylase b (97 kDa),
bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), and carbonic
anhydrase (31 kDa).
Analytical Gel Chromatography--
For the analytical gel
chromatography experiments, a Superdex-200 HR 10/30 column and FPLC
equipment (Amersham Pharmacia Biotech, Uppsala, Sweden) were employed.
The column was equilibrated with TKMD buffer (50 mM Tris,
72 mM KCl, 7.2 mM MgSO4, 0.2 mM DTT, pH 7.5) at room temperature, with or without a
ligand, and 100 µl of RKPK samples were loaded onto the column.
Fractions of 0.25 ml were collected, and PK activity was measured using
the method described above. The following proteins were used for column
calibration: catalase (240 kDa), aldolase (158 kDa), and albumin (66.2 kDa) (calibration proteins II for gel chromatography; Combithek,
Boehringer Mannheim).
Circular Dichroism--
Far-UV CD spectra of T340M rRKPK and
wild type rRKPK were measured with an Aviv 62 DS circular dichroism
spectrometer. rRKPK solutions of 0.6 mg/ml and T340M rRKPK of 1.0 mg/ml
were measured in a fused quartz cuvette with a pathlength of 0.1 cm.
Each spectrum was recorded in the region from 190 nm to 260 nm with a
0.5-nm increment and 1-s interval. For each sample, three repetitive scans were obtained and averaged.
Velocity Sedimentation--
Velocity sedimentation experiments
were performed in a Beckman OptimaTM XL-A analytical
ultracentrifuge (Palo Alto, CA) with an An-60Ti rotor, equipped with
absorption optics. The experiments were carried out at 30,000 rpm and
23 °C. Data were collected in the continuous mode with a radial
stepsize of 0.003 cm, at 8-min intervals. Data were collected, on
400-µl samples loaded into double sector centerpieces, at 230 nm for
rRKPK concentrations of 0.025 mg/ml and 0.050 mg/ml, and all other data
were collected at 280 nm. Weight-average sedimentation coefficients
were obtained by employing the Beckman data analysis software.
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RESULTS |
Purification of T340M rRKPK--
The T340M rRKPK was purified in
order to assess the kinetic and physical properties, employing the
protocol established for rRKPK and rRMPK (5, 13).2 The
purity of the recombinant T340M RKPK was approximately 95% homogeneous
based on a densitometric analysis of the SDS-polyacrylamide gel.
Typically, 1 liter of culture results in 3 mg of purified T340M rRKPK,
which is 2 times the yield of wild type rRKPK.2 Similar to
the wild type rRKPK, the activity of T340M rRKPK is unstable. It is
essential to carry out the purification method quickly. Generally, the
sample should be dialyzing against buffer C within 10 h after cell
disruption.
Kinetic Characterization of T340M rRKPK--
Steady state kinetics
obtained for both the wild type and T340M rRKPK, as a function of
effectors (data not shown for wild type), with PEP as a variable
substrate are shown in Fig. 2. Parameters derived from these steady state kinetics are summarized in Table I. In the absence of effectors, the
saturation curves of the wild type and T340M rRKPK are significantly
different. The value for Kapp,PEP has increased
from 0.16 mM for the wild type to 5.8 mM for
the T340M rRKPK mutant, a more than 30-fold increase. The apparent
cooperativity, quantitated by the Hill coefficient (n), was
not significantly affected by the mutation. Recent reports showed that
the interactions of PEP and Mg2+ with PK is coupled (19,
20). In view of the significant change in the value of
Kapp,PEP, the effect of Mg2+
concentration was studied by monitoring PK activity at fixed concentrations of 2 mM ADP and 20 mM PEP and
varying concentrations of MgSO4. Activity of T340M rRKPK
reached its maximum value at about 6 mM MgSO4,
after which there was no observable increase in activity with
increasing concentration of MgSO4. Thus, it can be
concluded that the kinetic measurements were conducted in the presence
of saturating concentration of MgSO4.

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Fig. 2.
Kinetic characterization of T340M rRKPK
titrated with PEP. Figure shows results for T340M rRKPK in the
absence of effectors ( ) or in the presence of 2 mM Phe
( ) or 2 mM Phe and 10 µM FBP ( ); and in
the presence of 100 µM FBP ( ) or 400 µM
FBP ( ). Wild type rRKPK titration in the absence of effectors ( ).
The lines through the data sets are the result of nonlinear fitting to
Equation 1. The kinetic parameters resulting from the fittings are
shown in Table I. The ordinates on the left and
right are for the T340M mutant and wild type PK,
respectively, and are shown in nanomoles of product/min.
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The presence of the inhibitor Phe (2 mM) led to an increase
in Kapp,PEP for both wild type and T340M rRKPK.
The increase in values for Kapp,PEP is 19- and
4-fold in wild type and T340M mutant, respectively. There is no
significant difference in the apparent cooperativity between wild type
and T340M rRKPK in the presence of Phe. The presence of both Phe (2 mM) and the activator FBP (10 µM) decreases
the apparent cooperativity and the value for Kapp,PEP for both wild type and T340M rRKPK. For
the wild type enzyme, the inhibitory effect of Phe is more than
reversed by 10 µM FBP, indicated by a smaller
Kapp,PEP and Hill coefficient as compared with
wild type in the absence of effectors. In the T340M rRKPK mutant,
however, the presence of 10 µM FBP only partially reverses the inhibitory effect of Phe. The T340M mutation decreases kcat by at least 2-fold under all three
conditions tested. The effect of FBP alone was tested. The activity of
T340M rRKPK increased with increasing concentration of FBP, as shown in
Fig. 3. The normalized data were fitted
to Equation 1. Values of 50 µM and 1.3 were obtained for
Kapp,FBP and n, respectively. Maximum
stimulation of activity was apparently achieved by 100 µM
FBP. Effect of FBP on Kapp,PEP was tested at two
ligand concentrations of 100 and 400 µM. Results are
shown in Fig. 2 and Table I. The values of Kapp,PEP and kcat remain
at approximately 1.8 mM and 100 s
1,
respectively, despite a 4-fold increase in FBP concentration. These
results imply that under these experimental conditions this mutant is
operating at the maximum velocity of which this enzyme is capable.

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Fig. 3.
FBP activation of T340M rRKPK. The
activity was monitored at fixed concentrations of 2 mM ADP
and 5 mM PEP in the presence of varying concentrations of
FBP. The enzyme activity in the absence of FBP was used as the
reference. The ordinate is in the unit of
(VFBP/Vo 1) where
VFBP and Vo are the
activities in the presence and absence of FBP, respectively.
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Steady state kinetics of wild type and T340M rRKPK as a function of ADP
concentration are shown in Fig. 4. The
fixed concentration of PEP was 20 mM and 2 mM
for experiments pertaining to the T340M mutant and the wild type
enzyme, respectively. Parameters derived from these measurements are
summarized in Table II. The
kcat determined with ADP as a variable substrate
is lower than that in the PEP titration (Table I). This is probably
because at 20 mM PEP one has not reached substrate
saturation for T340M rRKPK. These data indicate that the T340M mutation
does not significantly affect the apparent kinetic parameters with
respect to ADP as a substrate. A combination of all the steady state
kinetic data implies that the T340M mutation apparently renders the
enzyme less active with significantly lower affinity for PEP without
perturbing ADP binding.

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Fig. 4.
Kinetic properties of the wild type rRKPK
( ;2), and the T340M rRKPK ( ). ADP was the variable
substrate in the presence of fixed 2 mM and 20 mM PEP for wild type and T340M rRKPK, respectively. The
lines through the data sets are the results of nonlinear fitting to
Equation 1. The kinetic parameters resulting from the fittings are
shown in Table II. The ordinate is shown in nanomoles of
product/min.
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Circular Dichroism--
The secondary structure of T340M rRKPK and
wild type rRKPK were monitored by CD. There is no observable difference
in molar ellipticity under the experimental conditions as shown in Fig. 5. The identity of the CD spectra for
these two proteins indicates that the T340M mutant is folded into a
similar motif as the wild type enzyme as determined by this
spectroscopic technique.

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Fig. 5.
CD spectra in the far-UV region of wild type
rRKPK and T340M rRKPK in TKMD at 23 °C as a function of protein
concentration. Figure shows results for wild type rRKPK at 0.6 mg/ml ( ) and T340M rRKPK at 1.0 mg/ml ( ).
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Sedimentation Velocity--
Wild type rRKPK undergoes
self-association with a stoichiometry of 2D
T (13).2
The association-dissociation occurs at the 1,2 subunit interface (Fig.
1). This is based on the fact that the sequence difference between the
rRMPK, which is a stable tetramer, and the rRKPK, which undergoes
self-association, is at the 1,2 subunit interface. Moreover, the change
in accessible surface area upon subunit assembly at the 1,2 subunit
interface is about half of that of the 1,4 subunit interface. Thus, the
expected energetics of interfacial interaction along the 1,2 subunit
interface should be substantially less than that of the 1,4 interface.
However, the T340M mutation is located at the 1,4 subunit interface,
and an effect on the energetics at the 1,4 subunit interface might be
observed. A weakening in the energetics along the 1,4 subunit interface
might change the stoichiometry of the self-association reaction from
dimerization of dimers to tetramerization of monomers. The
stoichiometry of the self-association reaction can be evaluated by
applying the Gilbert theory to the sedimentation data (21). To describe
the self-association of T340M rRKPK quantitatively, the ability to aggregate was monitored by sedimentation velocity. Weight-average sedimentation coefficients of rRKPK were determined within the protein
concentration range of 0.025-1.5 mg/ml at pH 7.5 and 23 °C in TKMD
buffer. As expected for a system undergoing self-association, the
weight-average sedimentation coefficient is a function of protein
concentration, as depicted in Fig. 6. At
higher protein concentration, the weight-average sedimentation
coefficient converges to a value greater than 10 S. A similar value was
obtained for the tetrameric rRMPK and rRKPK (5, 13).2
Extrapolation of a double reciprocal plot of the weight-average sedimentation coefficient versus concentration to infinite
concentration yielded a value of 10.50 S for the largest aggregate of
this T340M mutant. The absorption optics of the analytical
ultracentrifuge places the lower limit of PK that can be monitored at
2.5 µg/ml, as shown in Fig. 6. Further analysis of the sedimentation
data was conducted to define the stoichiometry and equilibrium
constants of the self-association reaction because weight average
sedimentation coefficient is defined by Equation 2.
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(Eq. 2)
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sio and
Ci are the intrinsic sedimentation coefficient and
concentration of the ith species, respectively. The
relationship between
and total
protein concentration is a function of stoichiometry and equilibrium
constants and is related by Equation 3.
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(Eq. 3)
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gi is the hydrodynamic non-ideality term,
Ki is the association constant governing the
formation of i-mer from monomer and
K1 = 1, by definition, and
C1 is the monomer concentration. Fitting the
data of
versus C requires an estimation
of values of sio. The
s20,w values of the monomer and dimer
can be calculated employing Equation 4.
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(Eq. 4)
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The calculated sedimentation coefficients, based on a tetramer of
10.5 S, are 4.16 S and 6.61 S for the monomer and dimer, respectively.
This relationship assumes spherical symmetry for all species (22,
23).

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Fig. 6.
Weight-average sedimentation coefficients
( 20,w) of T340M rRKPK as a
function of total protein concentration in TKMD buffer at 23 °C
( ). The lines are least-square fitting to the experimental data
according to the models described in Table IV: model I (- - -); model
II (------); model III (- - -); and model II fitted to wild type
rRKPK from Footnote 2 (- -). Residuals are plotted as the observed
values minus the calculated values according to model I (×), model II
( ), and model III ( ).
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The concentration dependence of the weight-average sedimentation
coefficient was fitted to different modes of association using the
calculated s20,w values for the monomer
and dimer, and the experimentally determined
s20,w value of the tetramer. The results
of the least-squares fittings are described in Table
III. The calculated data employing model
I and II fit equally well to the experimental data. Fitting to model III, however, results in non-random residuals, which excludes model III
as a possible model.
Expressing the reaction boundary of a system in rapid dynamic
equilibrium as weight-average sedimentation coefficient can result in
the loss of valuable information. The shape of the sedimenting boundary
can be very diagnostic for detecting the mode of association according
to the Gilbert theory, which predicts a single sedimenting peak for
macromolecular dimerization (n = 2) and resolution into bimodal sedimentation profiles for higher order polymerization reactions (n
3). The appearance of bimodality in the
higher-order polymerization reaction is a function of the association
constants and total macromolecular concentration in the plateau
region.
Comparing the simulated and experimental sedimentation patterns can
provide diagnostic information in support of a model describing a
self-association equilibrium (21). Sedimentation velocity profiles were
simulated based on a method developed by Cox (24) and adopted by this
laboratory in earlier studies (25-27). The simulated profiles describe
a derivative of a sedimentation boundary at a fixed sedimentation time
and solute concentration, employing the fitted association constant and
values for s20,w for models I and
II.
Model I fits the
20,w
versus C data in Fig. 6 to a monomer-tetramer mode of association.
The simulated sedimentation profiles for two different protein loading
concentrations are shown in Fig.
7A. Because the stoichiometry
of polymer formation is 4 (n = 4), the observed
bimodality in Fig. 7A is consistent with the Gilbert theory.
Model II fits the
20,w
versus C data in Fig. 6 to a dimer-tetramer mode of association.
The dimerization constant derived from data analysis of Fig. 6 and s20,w values of the dimer and tetramer
were used to simulate a sedimentation pattern for two different protein
loading concentrations (Fig. 7B). Each of the simulated
sedimentation pattern exhibits a single sedimenting peak as predicted
by the Gilbert theory for a macromolecular dimerization
(n = 2).

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Fig. 7.
Simulated sedimentation velocity patterns of
rRKPK at 0.05 mg/ml (- -, sedimentation time of 10,650 s), and 0.20 mg/ml (------, sedimentation time of 10,970 s). A sharp initial
boundary was set at a radial distance of 6.1 cm for the 0.05 mg/ml
sample, and 6.13 for the 0.20 mg/ml sample. The ordinate is
the concentration gradient in milligrams/ml/cm, and the
abscissa is the radial position in centimeters from the
center of rotation. Simulation to model I of Table III is shown in
A, model II of Table III is shown in B, and
C represents the experimental derivatives of sedimentation
patterns at 0.05 mg/ml ( ) and 0.20 mg/ml ( ) of T340M rRKPK, after
sedimentation times of 10,650 and 10,970 s, respectively.
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Derivatives of experimental sedimentation patterns, at the protein
loading concentrations and sedimentation times used in the simulations,
are plotted in Fig. 7C. It is evident, based on the shape,
dependence on protein loading concentration, and amplitude on the
ordinate, that the experimental data resemble Fig. 7B best.
Therefore, it can be concluded that the mode of association is not
affected by the T340M mutation and remains as a dimerization of dimers
to tetramer. The fitted equilibrium constant for this mutant is
220 ± 20 ml/mg as compared with 28 ± 3 ml/mg determined for
the wild type rRKPK.
Analytical Gel Chromatography--
In an earlier report,
analytical gel chromatography was employed to show that wild type rRKPK
undergoes self-association and that the presence of metabolites
significantly affects the equilibrium (13).2 The same
approach was used to monitor changes in molecular size of T340M rRKPK
as a function of protein concentration and Phe concentration.
The elution profile of T340M rRKPK at 50 µg/ml, as shown in Fig.
8, is identical to that of tetrameric
rRKPK and rRMPK (data not shown), with respect to both elution volume
and shape of the profile (13).2 This indicates that T340M
rRKPK is predominantly tetrameric at this concentration, which is about
10-fold higher than the dissociation constant for the dimer-tetramer
reaction. The elution volume of wild type rRKPK at 12 µg/ml implies
the presence of a molecule substantially less than tetrameric PK. In
accordance to a dimerization constant of 28 ml/mg (13),2
then rRKPK should exist predominantly as dimer at this protein concentration. In contrast, at 2.5 µg/ml the T340M mutant exhibits an
elution volume and profile similar to that of tetrameric rRMPK (Fig.
8). This observation implies that the T340M mutant exhibits a higher
propensity to self-association, a conclusion in complete agreement with
the sedimentation data. The self-association of wild type rRKPK is
linked to the binding of Phe, such that Phe binding shifts the
equilibrium to the dimeric form.2 The elution profile of
T340M rRKPK at 10 µg/ml in the presence of 2 mM Phe (Fig.
8) or 12 mM Phe (data not shown), indicates that the mutant
remains predominantly tetrameric. The lack of response to Phe in the
analytical gel chromatography experiments is not the result of a
decreased affinity of T340M rRKPK for Phe, inasmuch as the kinetic
properties are responsive to 2 mM Phe (Fig.
2). Therefore, it can be concluded from
these experiments that the T340M mutation at the 1,4 subunit interface
results in significant enhancement in the energetics of subunit
assembly at the 1,2 subunit interface.

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Fig. 8.
Effects of ligands on the elution profile of
wild type rRKPK and T340 M rRKPK. Figures shows results for T340M
rRKPK at 50 µg/ml ( ) and 2.5 µg/ml ( ), and at 10 µg/ml in
the presence of 2 mM Phe ( ); and wild type rRKPK at 12 µg/ml in the absence of effectors ( ). The lines through the data
are drawn to indicate the trend of the data.
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DISCUSSION |
The major observations of this study are that T340M mutation
exerts a dominant effect on mammalian PK by lowering the catalytic efficiency and apparent affinity for PEP, and a decrease in
responsiveness to the presence of effectors while exhibiting no
apparent effect on ADP binding. Furthermore, the effects of T340M
mutation are propagated through long range communication to the active
site and the 1,2 subunit interface which are ~20 Å and ~45 Å away, respectively. Thus, these results provide the first direct
evidence to establish the communication network between the active site and both subunit interfaces.
Mutation of this highly conserved threonine to methionine in rRKPK
(this study), rRMPK (5), and human erythrocyte and liver PK isozymes
(7-9) results in a similar observation of decrease in enzyme activity.
These results provide support for the hypothesis that the molecular
mechanism of regulation in PK is conserved. The verification of this
assumption significantly increases the efficiency of selecting
functionally important structural elements. The information embedded in
human erythrocyte PK variants can be revealed by studying the
equivalent mutants of the other isozymic forms of PK. The sites of
mutation and the nature of amino acid substitution reported in these
human genetic variants can serve as leads in probing the molecular
mechanism of allosteric regulation in mammalian PK.
The T340M mutation significantly alters the kinetic properties of the
enzyme. By comparing the kinetic behavior of the wild type and T340M
rRKPK, there is an approximately 50% decrease in the value of
kcat. The effect of mutation on the steady state affinity for PEP is based on the observed perturbation on the values of
Kapp,PEP. Although one may question the validity
in equating these kinetically determined Kapp
parameters to actual equilibrium binding constants in rRKPK, it was
shown by independently determined ligand binding constants that the
Kapp parameters are reasonable approximation of
binding constants in RMPK (26-29). It is most interesting to note that
the same effects of T340M mutation were observed on the kinetic
behavior of the other form of PK, the rRMPK isozyme (5). Thus, the
sequence differences between the two isozymes along the 1,2 subunit
interface do not alter the dominating, negative effects of the T340M
mutation. These effects are a decrease in catalytic efficiency,
decrease in apparent affinity for PEP, and decreased responsiveness to allosteric effectors.
An important feature of allosteric regulation is the communication
between ligand binding sites and subunits of an oligomeric enzyme such
as PK. An outstanding advantage of rRKPK to elucidate the communication
network is its propensity to undergo a dynamic association-dissociation
reaction. The change in the energetics of subunit assembly can be
employed to reflect a communication via this subunit interface
(13).2 In wild type rRKPK, it was shown that binding of all
metabolites communicates through the 1,2 subunit interface. From the
results of both analytical gel chromatography and sedimentation
velocity experiments, it was apparent that the T340M mutation shifts
the equilibrium in favor of the tetrameric form. Thus, an obvious conclusion is that there is communication between residue 340 and the
1,2 subunit interface, although the exact mechanism of communication
has to be defined. The mechanism could involve a direct pathway between
these two sites, or it might be indirect through a global structural
perturbation by the T340M mutation. It is interesting to note that
analogous to the observed correlation between subunit assembly and
metabolite binding, a possible linkage can be considered between the
self-association equilibrium and the effect of Phe on the kinetic
behavior of the T340M mutant. In wild type rRKPK, the kinetic behavior
is more responsive to Phe and the presence of this ligand significantly
shifts the subunit assembly equilibrium toward the dimeric form.
However, in the presence of up to 12 mM Phe, the T340M
rRKPK remains predominantly tetrameric. These results indicate that the
T340M mutation in rRKPK affects the intrinsic properties of the 1,2 subunit interface, and there is an apparent correlation between the
decrease in responsiveness to Phe in enzyme kinetics and subunit
assembly equilibrium, namely less response with a decreased ability to
shift the subunit assembly equilibrium. Residue 340 not only
communicates with the 1,2 subunit interface, there is also
communication between residue 340 and the active site approximately 20 Å away. This conclusion is supported by the observation that the
catalytic efficiency of the T340M mutant is significantly decreased.
Communication between the active site and the 1,2 subunit interface was
established for the wild type rRKPK (13).2 Studies in this
report identify the communication between the T340M mutation at the 1,4 subunit interface and the 1,2 subunit interface. This completes a
triangulation of communication network within the rRKPK subunit between
the T340M mutation at the 1,4 subunit interface, the 1,2 subunit
interface, and the active site. This is the first report that
identifies the communication between these functionally important
structural elements in PK and in turn addresses a fundamental issue in
elucidating the regulatory mechanism of PK by identifying the network
of communication between functionally important sites, such as ligand
binding sites and subunit interfaces.
Results from small angle neutron scattering experiments and computation
modeling led to a proposal that the conversion of inactive RMPK to the
active state is associated with a rotation of the B domain toward the A
domain along the 1,4 subunit interface (31). As a consequence of this
domain rotation, one of the new contact sites involves residue 340. Thus, the observed perturbation of kinetics and communication pathway
reported in this study is consistent with the earlier proposal. Being
encouraged by these consistent results, it is useful to examine the
structure of RMPK determined by Larsen et al. (11) in order
to develop a model for the transmission of information between the 1,4 subunit interface and the active site.
The model indicates that the closed cleft conformation of the active
state is stabilized by intersubunit electrostatic interactions between
Asp-177 in the B domain of subunit 1 with Arg-341 of the A domain in
the subunit across the 1,4 subunit interface. Stabilization of the
active state by an electrostatic interaction between Arg-341 and
Asp-177 is consistent with results of a study of RMPK that indicated a
loss of activity in the presence of high salt concentrations (32).
Arg-341 is part of
-helix 12, which includes residues 341-353, that
constitutes part of the 1,4 subunit interface. It is possible that a
minor change, such as a T340M mutation, at the 1,4 subunit interface is
propagated through helix 12, resulting in a perturbation of the proper
positioning of its neighboring Arg-341 with Asp-177, resulting in the
destabilization of the active state. Asp-177, Arg-341, and Thr-340 are
conserved residues in all known PK sequences (10). This model is
consistent with the kinetic observations of the T340M mutants in rRKPK,
rRMPK, and human erythrocyte PK (5, 7-9) that the T340M mutation in
all three PK isozymes lead to the same net result, namely significant deficiency in PK activity. Of course, the validity of this proposed model awaits the high resolution structural information of this mutant
and further experimental results of mutants and solution biophysical
characterizations of these mutants. Nevertheless, it is interesting to
note that Mattevi et al. (33) identified the equivalent of
residue 341 in E. coli PK as playing a role in intersubunit
salt bridge formation to stabilize the T-state enzyme. Alternatively, a
communication pathway between residue 340 and active site may involve
transmission of information between residue 340 and the 1,2 subunit
interface, which in turn communicates to the active site. A
communication between the 1,2 subunit interface and active site has
been demonstrated in an earlier study (13).2 These two
models can rationalize part of the communication network that couple
different locations within the PK tetramer.
The significance of this mutation in affecting the molecular mechanism
of regulation has yet to be determined, inasmuch as allosteric
regulation is the net consequence of linked functions in which the
binding affinity of one ligand is linked to that of another ligand and
structural equilibria as formulated mathematically by Wyman (12). As a
consequence of these linkages, the observed fitted constants are
apparent parameters and can be distinct from their intrinsic values. To
assess the actual effect of the T340M mutation on the regulatory
mechanism at the molecular level, it is imperative to identify the
intrinsic properties of the various linked equilibria that are
affected. Further studies are being conducted to dissect these linked
equilibria.3