Hybrid Tetramers Reveal Elements of Cooperativity in
Escherichia coli D-3-Phosphoglycerate
Dehydrogenase*
Gregory A.
Grant
,
Zhiqin
Hu, and
Xiao
Lan
Xu
From the Departments of Molecular Biology and Pharmacology and
Medicine, Washington University School of Medicine, St. Louis, Missouri
63110
Received for publication, December 20, 2002, and in revised form, March 6, 2003
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ABSTRACT |
D-3-Phosphoglycerate
dehydrogenase from Escherichia coli is a tetramer of
identical subunits that is inhibited when L-serine binds at
allosteric sites between subunits. Co-expression of two genes, the
native gene containing a charge difference mutation and a gene
containing a mutation that eliminates serine binding, produces hybrid
tetramers that can be separated by ion exchange chromatography.
Activity in the hybrid tetramer with only a single intact serine
binding site is inhibited by ~58% with a Hill coefficient of 1. Thus, interaction at a single regulatory domain interface does not, in
itself, lead to the positive cooperativity of inhibition manifest in
the native enzyme. Tetramers with only two intact serine binding sites
purify as a mixture that displays a maximum inhibition level that is
less than that of native enzyme, suggesting the presence of a
population of tetramers that are unable to be fully inhibited.
Differential analysis of this mixture supports the conclusion that it
contains two forms of the tetramer. One form contains two intact serine
binding sites at the same interface and is not fully inhibitable. The
second form is a fully inhibitable population that has one serine
binding site at each interface. Overall, the hybrid tetramers show that
the positive cooperativity observed for serine binding is mediated
across the nucleotide binding domain interface, and the negative
cooperativity is mediated across the regulatory domain interface. That
is, they reveal a pattern in which the binding of serine at one
interface leads to negative cooperativity of binding of a subsequent
serine at the same interface and positive cooperativity of binding of a subsequent serine to the opposite interface. This trend is
propagated to subsequent binding sites in the tetramer such that the
negative cooperativity that is originally manifest at one interface is decreased by subsequent binding of ligand at the opposite interface.
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INTRODUCTION |
Escherichia coli D-3-phosphoglycerate
dehydrogenase (PGDH,1 EC
1.1.1.95) catalyzes the first committed step in L-serine
biosynthesis and is inhibited by the binding of L-serine to
an allosteric site (1-3). PGDH is a tetramer of identical subunits and
contains four catalytic sites as well as four effector sites (4). Each subunit is composed of three distinct domains, the substrate binding domain, the cofactor binding domain, and the serine binding or regulatory domain. Subunits make contact between adjacent regulatory domains at the regulatory domain interface (rdi) and between adjacent nucleotide binding domains at the nucleotide binding domain interface (ndi). The four catalytic sites are formed at the junction between the
substrate and cofactor binding domains, and the four effector sites are
formed at the two regulatory domain interfaces (Fig. 1). Hydrogen bonds to serine are
contributed by both subunits at the regulatory domain interface, and
serine appears to tether the two domains together through this hydrogen
bond network (4, 5). Two distinctive cooperative processes appear to
function in the inhibition of the enzyme by L-serine.
First, the inhibition of activity in response to serine binding
displays a sigmoidal behavior with a Hill coefficient of ~2.
Secondly, serine binding itself displays characteristics of both
positive and negative cooperativity (6). Furthermore, it has been
demonstrated (7, 8) that these two processes can be uncoupled by
specific amino acid residue mutations, producing mutant enzymes that
have lost their cooperativity of inhibition while maintaining their
cooperativity of serine binding. In addition, the degree of
cooperativity in serine binding is affected by the binding of NADH (9)
and the presence of phosphate ion (10) without an appreciable
concomitant effect on the cooperativity of inhibition. These
observations have led to the suggestion that the enzyme functions
through distinct structural pathways that can be uncoupled by mutations
or interactions with solvent constituents. Whereas these pathways may
share structural elements, their uncoupling by mutation also
demonstrates that they diverge structurally at some point. Thus, the
interaction of these pathways and the interplay of specific catalytic
and effector sites are of critical importance to understanding the regulatory mechanism.

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Fig. 1.
A diagram depicting the structure of the PGDH
tetramer. Each identical subunit contains three distinct domains,
the regulatory domain (r), the substrate binding domain
(s), and the nucleotide binding domain (n). The
active site is formed in a cleft between the substrate binding and
nucleotide binding domains. The tetramers contact each other at
interfaces between the regulatory domains and the nucleotide domains as
depicted. L-Serine, the negative effector, binds in the
regulatory domain interface.
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Because PGDH is composed of four identical subunits, a mutation made in
one subunit will occur symmetrically in all subunits. This property
makes it difficult to separate intra-subunit effects from inter-subunit
effects and to elucidate the precise relationship of one individual
site to another. In order to address this, it is necessary to produce
hetero-tetramers where the sites are modified asymmetrically.
Similar studies utilizing hybrid oligomers have been reported for
L-lactate dehydrogenase from Bifidobacterium
longum (11, 12) and for porcine fructose 1,6-biphosphatase (13).
Although these enzymes are homo-tetramers like PGDH, they differ
significantly in the arrangement of their substrate and effector
binding sites. B. longum L-lactate dehydrogenase
contains only two effector sites but has four active sites (14). The
effector sites in L-lactate dehydrogenase are formed by
residues from adjacent subunits, whereas the active sites are basically
contained within each subunit. Fructose 1,6-biphosphatase contains four
effector sites and four active sites (15). The substrate binding sites
in fructose 1,6-biphosphatase are at subunit interfaces, and the
effector sites are contained within individual subunits. In PGDH, it is
the effector sites that are found at subunit interfaces, and the
substrate sites for PGDH interact with residues from adjacent subunits.
In addition, the active site of PGDH is found in a distinct cleft
between two subunit domains that is predicted to open and close during
catalysis and remain closed in the inhibited state (4). Thus, there are distinct differences among these enzymes that could uniquely contribute to significant differences in their mode of action.
This study describes a method for producing and isolating specific
hybrid tetramers of PGDH and analyzes the properties of PGDH tetramers
containing zero, one, two, three, and four high-affinity serine binding sites.
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MATERIALS AND METHODS |
Protein was isolated and initially purified from bacteria
utilizing 5' AMP-Sepharose affinity chromatography as described previously (16, 17). Enzyme activity was determined by the change in
absorbance at 340 nm due to the conversion of NADH to NAD+
at pH 7.5 (18) using
-ketoglutarate as the substrate (19).
All PGDH constructs used in this study are based on PGDH 4CA, which is
a form of PGDH in which the 4 cysteine residues in the PGDH subunit
have been mutated to alanine residues. This construct behaves similarly
to native PGDH and has been widely used in previous studies (5-10).
The kcat and I0.5 for serine are
identical to those of the native enzyme, and the Km
for
-ketoglutarate is 5-fold higher than that of the native enzyme
(16). It is chosen so that it can be used later for introduction of
reporter groups at specific places on the subunit through new cysteine residues. For the sake of simplicity, this construct will be assumed as
the background in all subsequent descriptions and will not be referred
to specifically.
Construction of a Plasmid for the Co-expression of Two Distinct
PGDH Genes--
Hybrid or mixed tetramers were produced by expressing
two individual PGDH genes on the same plasmid under control of a single promoter. The plasmid pTrc PGDH (16) was used to
construct two intermediate plasmids, pTrc PGDH 1a and
pTrc PGDH 1b (Fig. 2). pTrc PGDH 1a was made by introducing an XhoI site
just after the stop codon and before the HindIII site in
pTrc PGDH. This gene contained a charge difference mutation
and a sequence tag as well as the native serine binding site residues.
Charge variation was introduced into PGDH by the mutation of four
surface glutamate residues (Glu-45, Glu-49, Glu-386, and Glu-387) to
arginine residues. A sequence tag was introduced by mutating a valine
residue at position 4 to an alanine residue. In this way, when hybrid
tetramers are formed from this subunit (pTrc PGDH 1a) and a
subunit containing the native valine residue (pTrc PGDH 1b),
the exact ratio of subunits in the tetramer can be determined
quantitatively by automated Edman sequencing. PTH-alanine and
PTH-valine yield similarly (Val/Ala = 0.939 ± 0.002) in
Edman sequencing so that integration of their peaks provides an
accurate estimate of the subunit ratios. This construct is called
4ER/V4A, and the Km, kcat,
and IC50 for L-serine for this construct are
essentially indistinguishable from those of the unmutated enzyme.

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Fig. 2.
Construction of the gene for production of
the hybrid tetramers. Each subunit is coded by a separate gene
that is expressed under the control of a single promoter. Appropriate
mutations are placed in the respective genes and then combined in
pTrc PGDH duo/serine for expression in E. coli.
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pTrc PGDH 1b was made by introducing an XhoI site
just before the NcoI site in pTrc PGDH. This gene
contained the mutation of the serine binding site, N364A. Asn-364 is
one of the three serine binding residues found in the regulatory
domains of PGDH. This mutation decreases the IC50 for
serine from 10 µM in the native enzyme to 48 mM in the mutant (5). This effectively renders the site
incapable of binding serine over the range of serine concentrations
used in this study.
Mutations were produced in either pTrc PGDH 1a or pTrc
PGDH 1b by PCR as described previously (16) in preparation for
production of pTrc PGDH duo/serine. pTrc
PGDH duo/serine was produced by placing the
XhoI/HindIII fragment from pTrc PGDH
1b into the XhoI/HindIII sites in pTrc
PGDH 1a. Simultaneous expression of both genes in pTrc PGDH
duo/serine was induced with
isopropyl-1-thio-
-D-galactopyranoside and isolated with
a 5' AMP-Sepharose affinity column as described previously
(17).
Purification of Charge-differentiated Hybrid
Tetramers--
Hybrid tetramers containing zero to four high-affinity
L-serine binding sites were produced by expression of
pTrc PGDH duo/serine. Mutant hybrid tetramers
were purified by chromatography on QAE-Sepharose in 1 mM
potassium phosphate buffer, pH 7.5, 60 mM KSCN and eluted with a linear gradient of NaCl from 0 to 0.5 M. Fractions
were pooled and dialyzed against appropriate buffers before further purification or analysis. Pools that were not well resolved into homogeneous tetramers were re-chromatographed on QAE-Sepharose using
the same conditions with a gradient from 0 to 0.3 M NaCl. The distribution of subunits within a pool was determined by automated Edman sequencing of the sequence tag region as described above.
Inhibition Analysis--
Serine inhibition plots were fit to the
Hill equation (20, 21),
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(Eq. 1)
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where Imax is the maximum inhibition, I is the
fractional inhibition, L is the concentration of ligand, n
is the Hill coefficient, and I0.5 is the inhibitor
concentration at half-maximal inhibition. Protein concentration
was determined by quantitative amino acid analysis.
Serine Binding Analysis--
Serine binding was measured by
equilibrium dialysis in 200-µl dialysis chambers (Sialomed, Inc.,
Columbia, MD) purchased from the Nest Group (Southborough, MA).
Dialysis was performed for 16 h with
L-3[H]serine in appropriate concentrations of
unlabeled L-serine. Cells were sampled in triplicate, and
the average of 10-min counts was used to calculate
concentrations of free and bound L-serine. The nominal PGDH
concentration was 5-10 µM tetramer in all binding experiments, and all binding was performed in the presence of 100 µM NADH. Serine binding data were fit to the Adair
equation (21) for one (Eq. 2), two (Eq. 3), three (Eq. 4), or four (Eq. 5) sites using Kaleidograph (Synergy Software) as described previously (6, 7).
The Adair equations in the form of dissociation constants are shown
below.
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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where Y is the fractional occupancy, L is the free
ligand concentration, and Ki values are the
stepwise Adair constants. Plots of Y versus free
serine concentration were constructed with Y = r, Y = r/2, Y = r/3, and Y = r/4 for tetramers
containing one, two, three, and four functional serine binding sites, respectively.
For the tetramer pool with two mutant sites, where three species are
present (see Fig. 3), the Adair equation was modified as shown below to
account for each species.
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(Eq. 6)
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Intrinsic site dissociation constants were calculated from the
Adair constants by using the following statistical relationships for a
molecule where n sites are available and where
Ki' values are the intrinsic dissociation constants
(20).
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(Eq. 7)
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(Eq. 8)
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(Eq. 9)
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RESULTS |
Recombination of Co-expressed Subunits--
The expression of
pTrc PDGH duo/serine is expected to produce a
mixture of mutated hybrid tetramers as depicted in Fig.
3. Single species should be produced for
hybrid tetramers containing zero, one, three, and four mutated serine
binding sites. On the other hand, three species would be expected for
the hybrid tetramer containing two mutated serine binding sites.
However, because of the binding site symmetry and the fact that serine
binds between subunits, two of these (the bottom two in the
middle column in Fig. 3) are expected to be functionally
equivalent because they contain one binding site at each interface.
Unless otherwise noted, these two forms were treated equivalently.

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Fig. 3.
Depiction of the expected products from
recombination of the expressed subunits. Top, depiction
of the arrangement of serine binding residues at the two regulatory
domain interfaces in the PGDH tetramer. The filled circle
represents a serine molecule. Each serine binds to His-344 and Asn-346
on one subunit and Asn-364 on the adjacent subunit. Two serine
molecules bind at each interface with 180° symmetry.
Bottom, the expected hybrid tetramers produced when a N364A
mutation is introduced into one of the genes (pTrc PGDH 1b
in Fig. 2). A normal subunit (containing Asn-364) is depicted as
n, and a mutated subunit (containing Ala-364) is depicted as
x. The relative charge arising from the 4ER mutation in
pTrc PGDH 1a is shown above the expected tetramer.
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Each group of tetramers will have different charge properties
corresponding to the number of 4ER/V4A subunits they contain. The
initial separation of the hybrid tetramers produced by the expression
of pTrc PGDH duo/serine is shown in Fig.
4. Peaks were pooled and analyzed by
Edman degradation. Pools that were heterogeneous with respect to
tetramer content as judged by the Val/Ala ratio were re-chromatographed
as described under "Materials and Methods" (data not shown). The
homogeneity of the final tetramer pools based on subunit distribution
is shown in Table I. The kinetic parameters presented in Table II are
similar for all hybrid tetramers, suggesting that the active sites are
not compromised by the various mutations used.

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Fig. 4.
Chromatogram of the initial DEAE-Sepharose
fractionation of hybrid tetramers from the expression of pTrc PGDH
duo/serine. Loading and elution conditions are described in the
text. Absorbance at 280 nm ( ) is plotted against fraction number.
The NaCl gradient is depicted by the solid line.
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Serine Inhibition of Hybrid Tetramer Activity--
The serine
inhibition profiles of the hybrid tetramers are shown in Fig.
5, and the results of fitting the data to
the Hill equation are presented in Table
III. The tetramer with four intact serine
binding sites produced the expected inhibition pattern, which is
similar to unmutated PGDH, where the Hill coefficient is ~2, and
inhibition of activity is >95%. The tetramer with only three intact
serine binding sites also displayed inhibition of activity of >95%
but displayed a diminished sensitivity to serine at intermediate
concentrations. The Hill coefficient was also reduced to a value of
~1.6. The tetramer pool with only two intact serine binding sites
produced a Hill coefficient of ~1.2 but showed even more reduction in
sensitivity to serine and a reduced total inhibition of activity of
~85-90%. The tetramer with only a single serine binding site
displayed a Hill coefficient of 1, but its activity is inhibited only
~58%. As expected, the tetramer with no intact serine binding sites
was not inhibited by serine.

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Fig. 5.
Serine inhibition profiles of the hybrid
tetramers. Fractional inhibition is plotted against serine
concentration. The symbols are experimental results, and the
solid lines are the data fit to the Hill equation. Results
of PGDH hybrid tetramers with four ( ), three ( ), two ( ), one
( ), and zero ( ) high-affinity serine binding site(s) are
shown.
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Table III
Serine inhibition parameters
Parameters were derived from fitting the data to the Hill equation.
n is the Hill coefficient, S0.5 is the
serine concentration at one-half the maximum inhibition level,
Imax is the maximum inhibition level, X2
represents the cumulative deviation between the data and the fit, and
R is the correlation coefficient.
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Serine Binding to the Hybrid Tetramers--
The serine binding
curves fitted to the appropriate Adair equations are presented in Fig.
6, and the derived dissociation constants
are presented in Table IV. Note that all
serine binding experiments were performed in the presence of saturating
NADH, which moderates the degree of cooperativity (9). The
stoichiometry of serine binding measured for these mutants correlates
well with the expected number of intact serine binding sites. The data
for tetramers with one, three, and four intact serine binding sites can
be fit satisfactorily with Adair equations derived for the respective
number of sites. A special case exists for the tetramer with only two
intact serine binding sites. As shown in Fig. 3, three species are
predicted statistically, and based on symmetry, at least two of these
are expected to be functionally equivalent. Attempts to fit the data to
a single Adair equation for two sites failed to produce a satisfactory
fit. However, when the fit was attempted for an Adair equation composed
of three successive two-site equations with equal weight, the fit shown
in Fig. 6 was achieved. This fit produced binding parameters (shown in
Table IV as three separate entries) that indicated that two of the
three predicted species were equivalent. This corresponds to the
predicted functional equivalency of two of the three species based on
symmetry considerations.

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Fig. 6.
Plots of serine binding data. Left
panel, plots of the number of mols of L-serine
(r) bound per tetramer versus the free serine
concentration determined by equilibrium dialysis. The
symbols are the experimental data, and the solid
lines are the fit of the data to the appropriate Adair equation as
indicated in the text. Results of PGDH hybrid tetramers with four
( ), three ( ), two ( ), and one ( ) high-affinity serine
binding site(s) are shown. Right panel, Scatchard plots of
the data shown on the left.
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Table IV
Serine binding parameters
Binding data are expressed as dissociation constants
(µM). Adair constants are denoted as
Ki and intrinsic binding constants as
Ki'.
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DISCUSSION |
A complete set of hetero-tetramers of PGDH have been produced and
isolated that differ sequentially in the number of functional serine
binding sites that they contain. This has been accomplished by
co-expression of two genes for PGDH in E. coli. One gene
contained the single serine binding site mutation, and the other gene
contained the native serine binding site as well as charge mutants on
the surface of the subunit and a sequence tag at the amino terminus of
the polypeptide chain. Upon expression and folding, the subunits combined to give the expected mixture of tetramers, which were then
separated on an ion exchange column by virtue of the number of
charge-mutated subunits each tetramer contained. The stoichiometry of
the hetero-tetramers was verified by automated Edman sequencing of the
sequence tag region.
The serine inhibition profiles of the hybrid tetramers display
distinctly different patterns. The activity of the tetramer containing
only a single intact serine binding site (three mutated sites) is
inhibited to only ~58%. The simplest explanation for this
observation is that the binding of a single effector molecule at one
rdi can effectively inhibit both active sites in the subunits that
contribute to that interface. The observation that the extent of
inhibition is slightly greater than 50% also suggests that there may
be a small influence on the activity of the catalytic sites in the
other two subunits as well. This presumably results from a
conformational change induced by the binding of the first serine
ligand. The Hill coefficient of this tetramer is 1, which also suggests
that interaction across this regulatory domain interface does not, in
itself, produce the cooperativity of inhibition of catalytic activity
seen in the native enzyme. However, this same interaction undoubtedly
contributes to cooperativity of binding of subsequent ligands in the
native enzyme. This interpretation is also consistent with the
observation that all of the other hybrid tetramer pools, all of which
contain tetramers that bind serine at both regulatory domain
interfaces, display positive cooperativity for serine inhibition.
Because this tetramer contains only a single serine binding site, no
cooperativity of serine binding is possible, and the data produce a
hyperbolic binding curve yielding a single dissociation constant.
The results for the tetramer species containing two and three intact
binding sites are particularly revealing. The pool containing tetramers
with only two intact serine binding sites (two mutant sites) displays a
maximum level of inhibition of activity that is less than that of
native enzyme. This suggests the presence of a population of tetramers
whose activity is unable to be fully inhibited. Indeed, the
distribution of mutant tetramers shown in Fig. 3 predicts that
one-third of the tetramers will bind serine at only one
regulatory domain interface. This species would be expected to behave
similarly to the tetramer with only a single serine binding site
discussed above because serine interaction occurs only at one
interface. That is, its activity should be ~58% inhibited at
maximum. Thus, if the activity of one-third of the species is inhibited
to ~58% of total, and that of two-thirds of the species is inhibited
~98%, the catalytic activity of the whole population would be
inhibited by ~85%, which is what is seen experimentally.
Fitting the binding data to three successive Adair equations for two
binding sites each produces a fit that partitions the species in a
one-third and two-thirds distribution of total as predicted. The
species predicted to bind serine at both regulatory domain interfaces
shows positive cooperativity for the second ligand. The species
predicted to have both intact serine binding sites at the same
regulatory domain interface solves with extreme negative cooperativity
for the second ligand. This is consistent with the hypothesis (6) that
the potential binding of two effector molecules at a single interface
could explain the negative cooperativity of ligand binding if a single
effector molecule was capable of stabilizing the association of the two
domains to the extent that it could exclude or partially exclude the
binding of the second ligand.
Fitting the serine inhibition profile of the pool with two intact
serine binding sites to two successive Hill equations weighted for the
predicted distribution of these species produces a fit (Fig.
7) in which the smaller population
(one-third) displays a Hill coefficient of <1 (0.67 ± 0.42) and
an I0.5 of 45 µM, and the larger population
(two-thirds) displays a Hill coefficient of >1 (1.4 ± 0.18) and
an I0.5 of 6 µM. The I0.5 for
this smaller population is quite close to the Kd (40 µM) determined for serine binding of the tetramer with
one intact serine binding site (see Table IV). The larger population
has a Hill coefficient indicating positive cooperativity and an
S0.5 within the range for the Kd values
determined for this population from serine binding. Thus, both the
multicomponent Adair equation and the multicomponent Hill equation
produce fits that indicate that the population at one-third of the
total species basically mimics the characteristics of the tetramer with
only a single intact serine binding site.

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Fig. 7.
Fitting the serine inhibition data for the
two-site species to two successive Hill equations. The symbols
( ) are the data from Fig. 5, and the fit is produced with two
successive Hill equations weighted in a one-third to a two-thirds ratio
as predicted in Fig. 3. The X2 value for the fit is
0.017265, and the correlation constant, R, is 0.99489. The
dashed line indicates the hyperbolic response to the dual
Hill equation (i.e., n = 1).
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The tetramer with three intact serine binding sites (one mutated site)
has a Hill coefficient of 1.6 and displays nearly complete inhibition
at higher serine concentrations. The serine binding parameters show the
expected positive cooperativity for the second ligand and negative
cooperativity for the third ligand. However, note that the negative
cooperativity for the third ligand, which binds at the same interface
as the first ligand, is not as extreme as that seen for the tetramer
with two intact serine sites at the same interface discussed above.
This suggests that the binding of the second ligand at the interface
opposite to that where the first ligand binds is exerting its own
positively cooperative effect on the opposite interface. Although the
dissociation constant for the third ligand is higher than that for the
first two, indicating negative cooperativity, it is significantly lower
than if it were not being influenced by an interaction at the opposite
regulatory domain interface. This apparent lessening of negative
cooperativity is seen to an even greater degree for the unmutated
tetramer in which all four sites interact with serine.
Taken together, these results reveal a pattern in which binding of
serine to a second site at the same interface is negatively cooperative, and binding of serine to a second site on the opposite interface is positively cooperative. Fig.
8 depicts this concept in diagrammatic
form. The positive cooperativity observed for serine binding is
mediated across the ndi and is a result of a conformational change
induced at the second interface that increases the binding affinity for
ligand at that interface. At the same time, binding at a particular
interface is negatively cooperative for additional ligand binding at
that interface but can be modified by interaction at the opposite
interface. In terms of the nomenclature used in Fig. 8,
binding at site 1 enhances affinity at site 2 but reduces affinity at
site 3. However, subsequent binding at site 2 moderates the negative
cooperativity at site 3 to the extent that measurable ligand binding
takes place. Similarly, binding to site 3 ameliorates the negative
effect that occupancy at site 2 exerts on site 4 so that measurable
binding also occurs at site 4.

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Fig. 8.
Schematic depiction of the influence that
serine binding to a particular binding site has on serine binding to
subsequent sites. The four serine binding sites are represented by
the filled circles labeled Ser. The order of binding deduced
from this study is indicated by numerals next to the binding
sites. The two binding sites at the top of the figure are at
the same rdi, and the two binding sites at the bottom of the
figure are at the opposite rdi as depicted in Fig. 1. The two
regulatory domain interfaces (rdi) are at opposite ends of
the tetramer separated by the nucleotide binding domain interface
(ndi). The influence that each binding event has on
subsequent binding events is depicted with arrows, and the
sign indicates that it has either a positive (+) or negative
( ) influence.
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Overall, the binding of a single serine at each interface produces
substantial inhibition of all four active sites in a positively cooperative manner. This appears to be the major factor in the regulation of the enzyme. Binding of additional serines has a lesser
effect but does incrementally increase the degree of inhibition obtained and incrementally decreases the subsequent dissociation constants (higher affinity). However, because two regulatory domain interfaces regulate four active sites, the binding of a second serine
at each interface may not play a significant role in the regulation of
the enzyme.
The picture of PGDH that is emerging from these and previous studies
suggests that there is a significant entropic element to the regulation
of this enzyme. PGDH has anecdotally been inferred to be a very
flexible tetramer because of the large amount of "open space" in
its crystal structure and the great difficulty that has been
encountered in obtaining stable crystals of the uninhibited enzyme.
This mobility has been shown to be most likely due to the movement of
rigid domains about flexible hinges between each of the three domains
in each subunit (7, 8, 22). Binding of serine at the rdi is predicted
to draw the domains together to produce a tetramer with diminished
freedom of inter-domain motion. The diminished freedom of motion
experienced at one regulatory domain interface translates to the
opposite regulatory domain interface as a result of producing a more
rigid tetramer overall. The increasing rigidity results in an inability
of the enzyme to turn over rather than a decrease in the ability of
substrates to bind, consistent with the observation that it is largely
a "V"-type enzyme (2).
This investigation has allowed the determination of the order of
binding of effector molecules to PGDH and provided evidence to discern
the characteristics of individual effector binding sites and how they
directly effect the characteristics of the other binding sites and the
active sites. Additional investigations with hybrid tetramers
containing other mutations will continue to define the site to site
relationship in this enzyme as well as the structural elements that
mediate the process.
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FOOTNOTES |
*
Supported by National Institutes of Health Grant GM 56676.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Biology and Pharmacology, Box 8103, Washington University School of
Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-3367; Fax: 314-362-4698; E-mail ggrant@pcg.wustl.edu.
Published, JBC Papers in Press, March 18, 2003, DOI 10.1074/jbc.M213050200
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ABBREVIATIONS |
The abbreviations used are:
PGDH, D-3-phosphoglycerate dehydrogenase;
rdi, regulatory domain
interface;
ndi, nucleotide domain interface.
 |
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