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INTRODUCTION |
D-3-Phosphoglycerate Dehydrogenase
(PGDH,1 EC 1.1.1.95) from
Escherichia coli is a tetramer of identical subunits (1), the enzymatic activity of which is regulated in an allosteric manner by
L-serine, the end-product of its metabolic pathway (2-4). The crystal structure clearly shows that each subunit is made up of
three distinct domains (1). These are referred to as the regulatory (or
serine binding), substrate binding, and nucleotide binding domains.
Each regulatory domain forms an interface with another regulatory
domain from an adjacent subunit. The same is true for the nucleotide
binding domain so that there are two regulatory domain interfaces and
two nucleotide binding domain interfaces in each tetramer. The
substrate binding domain lies between the other two domains and does
not form an intersubunit contact. The regulatory domain is linked to
the substrate binding domain by a single strand of polypeptide that
contains a single Gly-Gly sequence approximately midway between the two
domains. The function of this Gly-Gly sequence has been investigated by
site-directed mutagenesis (5), and the data are consistent with its
functioning as a hinge region between the two domains involved in the
transmission of the effect of serine binding to the active site.
The crystal structure of PGDH, which has only been solved for the
enzyme with L-serine bound (1), reveals only a limited number of contacts between the regulatory and substrate binding domains. In this study, the nature of these contacts has been investigated by site-directed mutagenesis, and the results indicate that they play a key role in the cooperativity of L-serine
binding and L-serine inhibition.
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MATERIALS AND METHODS |
Mutants of PGDH were produced, expressed in E. coli,
and isolated as described previously (6, 7). Catalytic activity was
determined at 25° C in 20 mM Tris buffer at pH 7.5 using
-ketoglutarate as the substrate and by monitoring the decrease in
absorbance of NADH at 340 nm (8, 9). Protein concentration was
determined by the Bradford method (10, 11) and by quantitative amino acid analysis for the equilibrium dialysis binding experiments. All
mutations are constructed in PGDH4C/A to maintain
consistency with previous studies (5, 6, 10, 12). PGDH4C/A
is a form of the enzyme where the four native cysteine residues in each
subunit have been converted to alanine (10). The IC50 value for L-serine is that concentration of serine that produces
a 50% inhibition of the enzyme activity. Kinetic parameters were
determined using direct linear plots (13). Enzyme homogeneity was
judged by SDS gels. Figs. 1-4 were produced with Molscript
(14).
The oligomeric association state of the mutants was monitored by
intrinsic fluorescence and serine binding. Subunit dissociation results
in a shift in fluorescence from 340 to 360 nm (10, 15) and a loss of
serine binding (16). All mutants in this study that were recovered with
activity maintained an emission maximum at 340 nm and retained their
ability to bind serine, indicating an intact association of subunits.
Equilibrium dialysis was performed in 500-µl dialysis cartridges
obtained from Sialomed, Inc. (Columbia, MD). Dialysis was performed for
16 h with L-[3H]serine as a tracer in
appropriate concentrations of unlabeled L-serine. Cells
were sampled in triplicate, and the average of 10-min counts were used
to calculate concentrations of free and bound L-serine. The
nominal PGDH concentration was 5 µM tetramer in all
binding experiments.
Serine inhibition plots were fit to the Hill equation (12) (Equation 1),
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(Eq. 1)
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and serine binding data were fit to the Adair equation (12)
(Equation 2),
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(Eq. 2)
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or the equation for independent binding sites (Equation 3),
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(Eq. 3)
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using Kaleidograph (Synergy Software). Y is the
fractional occupancy or inhibition, r is mol of ligand bound
per mol of acceptor, p is the number of binding sites, and
n is the Hill coefficient. Intrinsic site dissociation
constants were calculated from the Adair constants by using the
following statistical relationships for a molecule where n
sites are occupied at maximal binding and where Ki'
are the intrinsic dissociation constants (17). n = 2 sites: K1' = 2K1,
K2' = K2/2;
n = 3 sites: K1' = 3K1, K2' = K2, K3' = K3/3; n = 4 sites:
K1' = 4K1,
K2' = 3K2/2,
K3' = 2K3/3, K4' = K4/4.
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RESULTS |
Description of the Regulatory Domain-Substrate Binding Domain
Interface--
Fig. 1 shows an
-carbon chain diagram of a subunit of PGDH. The nucleotide
binding domain (bottom) is linked to the regulatory domain
(top) by way of a helix that extends along the body of the
substrate binding domain (shaded dark). The regulatory and substrate binding domains are covalently linked by a single strand of
polypeptide, and the substrate and nucleotide binding domains are
covalently linked by two strands of polypeptide. The only apparent interaction between the regulatory and substrate binding domains are hydrogen bonds between the guanidino groups of Arg-339, Arg-405, and Arg-407 on the regulatory domain and main chain carbonyl groups on the substrate binding domain (Figs.
2 and 3).
Specifically, the guanidino group of Arg-339 forms hydrogen bonds with
the main chain carbonyls of Leu-332 and Asn-318, the guanidino group of Arg-407 forms a hydrogen bond with the main chain carbonyl of Ser-316,
and the guanidino group of Arg-405 forms a hydrogen bond with the main
chain carbonyl of Arg-97 and Leu-76. In addition, Arg-339 participates
in a hydrogen bonding network with Tyr-410 (the C-terminal residue of
the protein), Arg-338, Glu-387, and Ala-385 (Fig.
4). Specifically, the ring hydroxyl of
Tyr-410 is within hydrogen bonding distance of the guanidino group of
Arg-339 and the main chain carbonyl of Gly-336. The C-terminal carboxyl of Tyr-410 is within hydrogen bonding distance of the guanidino group
and the main chain amino group of Arg-338. The guanidino group of
Arg-338 is also within hydrogen bonding distance of the side chain
carboxyl of Glu-387 and the main chain carbonyl of Ala-385. This
network, along with the interaction of Arg-339 with Leu-332 and
Asn-318, completes a bridge across the loop containing Gly-336 and
Gly-337 (Figs. 2 and 3), which has been shown to be functional in
transmitting the effect of serine binding to the active site (5).

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Fig. 1.
-Carbon tracing of a subunit of
PGDH. The regulatory domain is at the top of the
figure, the substrate binding domain in the middle, and the
nucleotide binding domain at the bottom. The -helix that
runs from the nucleotide binding domain to the regulatory domain and
joins with the single strand connecting the regulatory domain to the
rest of the subunit is shaded dark. The subunit interfaces
are between adjacent regulatory domains (top right) and
adjacent nucleotide domains (bottom).
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Fig. 2.
-Carbon tracing of a regulatory
domain of PGDH and the adjacent polypeptide with which it
interacts. The regulatory domain is shaded light. The
polypeptide connecting to the -helix leading to the nucleotide
binding domain is shaded dark and extends from Gly-337
(G 337) to Asn-318 (N 318), which is at the
beginning of the helix. The side chains of residues described in the
text are depicted in ball and stick form. The potential
hydrogen bonding interactions are depicted with lines.
Nitrogens are shaded dark, carbons are shaded
medium, and oxygens are shaded lightest.
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Fig. 3.
An -carbon tracing
of a regulatory and substrate binding domain showing the
domain-spanning orientation of the side chains of Arg-405 (R
405), Arg-407 (R 407), and Arg-339 (R
339). The connecting polypeptide and the -helix from
the nucleotide binding domain are shaded dark as in Fig. 1.
The arginyl side chains and the main chain carbonyls with which they
potentially interact are shown in ball and stick
representation. Potential hydrogen bonds are shown with
lines. Nitrogens are shaded dark, carbons are
shaded medium, and oxygens are shaded
lightest.
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Fig. 4.
A close-up view of the residues shown at the
bottom of Fig. 2, which form a potential hydrogen
bonding network involving Arg-339 (R 339).
Potential hydrogen bonds are shown with lines, and the
hydrogen bonding distance is denoted in angstroms. Gly-336, which would
be behind the ring of Tyr-410 (Y 410) is not shown for
clarity. Nitrogens are shaded dark, carbons are shaded
medium, and oxygens are shaded lightest.
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Fig. 3 shows a view of the regulatory domain-substrate binding domain
interface looking along the
-sheet of the regulatory domain. This
clearly shows the nature of the trans-domain contacts made by the three
arginine residues that span the interface. Arg-405 is the only one that
interacts directly with the main body of the substrate binding domain.
The other two interact with the connecting polypeptide that starts at
the substrate binding domain-spanning
-helix and connects to the
main body of the regulatory domain.
Site-directed Mutagenesis--
The arginyl residues that span the
interface, Arg-405, -407, and -339, were converted to alanine
individually (Table I). R405A and R407A
both had a principle effect on the kcat of the enzyme, which resulted in a lowering of the kcat
and kcat/Km values by 1 and 2 orders of magnitude, respectively. In addition, both mutations produced
an enzyme that was more sensitive to serine and that displayed a
significant decrease in the Hill coefficient for cooperativity of
inhibition. The double mutant, R405A/R407A, appeared to have a profound
effect on the structure of the enzyme, resulting in the disruption of
the nucleotide binding site as judged by the protein's inability to
bind to the 5'-AMP affinity column. Activity and serine sensitivity
were restored by incorporating more polar residues at these sites
(R405N/R407N), but the loss in cooperativity of serine inhibition
remained.
R339A and R339N yielded low levels of protein that lacked sufficient
activity to be accurately measured. On the other hand, R339K produced
protein in good yield with a 10-fold reduction in
kcat, a small decrease in serine sensitivity,
and a reduced Hill coefficient.
Conversion of Arg-338 to Gly or removal of Tyr-410 (
Y410) from the C
terminus decreased serine sensitivity by 40- and 20-fold, respectively,
without a major effect on the
kcat/Km or the Hill
coefficient. Y410A displayed kinetic parameters similar to that of
Y410 and with a modest increase in serine sensitivity. E387A showed
little effect on any of the measured parameters.
The mutant that showed the greatest effect on the Hill coefficient,
R407A, was analyzed for its ability to bind serine by equilibrium
dialysis. The data were fit to the Adair equation for cooperative sites
(Fig. 5) as well as to the equation for equivalent, independent sites (Fig. 6).
The Adair equation was fit for four binding sites whereas the equation
for equivalent sites was fit for two total sites. The latter was
necessary because the equation for independent sites, unlike the Adair
equation, does not accommodate an analysis with less than total site
occupancy. Visual as well as statistical analysis (
2
value) indicates that the best fit is produced by the Adair equation, which can accommodate sites that are not independent. Both analyses indicate that there is a significant decrease in the intrinsic dissociation constant for the first site (Table
II). The Adair fit also indicates that
there is still a low level of positive cooperativity for binding to the
second site. This is consistent with an upward concavity at the
beginning of the Scatchard plot (not shown).

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Fig. 5.
Binding of L-serine to R407A
PGDH. The data are plotted as fractional occupancy of sites
(Y) versus free serine concentration
(µM). The solid line is the fit of the data to
the Adair equation for a molecule with four sites. The top
panel shows the full range of ligand concentration, and the
bottom panel expands the plot at low ligand
concentrations.
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Fig. 6.
Binding of L-serine to R407A
PGDH. The data are plotted as mol of ligand bound per mol of
tetrameric protein (r) versus free serine
concentration (µM). The solid line is the fit
to the equation for two independent sites. The top panel
shows the full range of ligand concentration, and the bottom
panel expands the plot at low ligand concentrations.
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Table II
Analysis of binding data
Binding data are expressed as dissociation constants. Adair constants
are denoted as Ki, and intrinsic site dissociation
constants are denoted as K'i.
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DISCUSSION |
Mutation of the arginyl residues spanning the regulatory
domain-substrate binding domain interface of PGDH produced profound effects on its sensitivity to serine, the cooperativity of serine inhibition, and in some cases, the apparent overall conformation of the
enzyme. Conversion of Arg-405 and Arg-407 to alanine, individually, produced similar effects that differed mainly in the extent of the
change. Although the values for kcat were
reduced by 1 and 2 orders of magnitude, respectively, a substantial
decrease in the Hill coefficient was also produced especially for
R407A. Serine binding measurements on R407A demonstrated that only two
of the four sites are occupied and that there is a substantial decrease in positive cooperativity among these sites. The lack of binding of the
last two sites is similar to that seen with the native enzyme. It has
been proposed (12) that occupancy of one site at the binding interface
closes the interface and precludes binding of the second molecule
within the same interface. That this appears to occur with greater ease
with this mutant is consistent with the notion that eliminating the
interaction mediated by the side chain of Arg-407 may ease a
conformational restraint so that the interface can close more easily in
response to the first ligand.
The fit to the Adair plot seems to be better than to that for a
rectangular hyperbola for independent sites, both by inspection and by
consideration of the values for
2. The positive
cooperativity of binding is also significantly reduced by this
mutation. Thus, although serine is still an effective inhibitor, the
degree of interaction between subunits appears to be diminished, and
the apparent reduction in positive cooperativity is consistent with a
significantly reduced Hill coefficient. In addition, the observation
that overall sensitivity to serine concentration increases while
cooperativity decreases reinforces the previous conclusion (12) that
simple inhibition (noncooperative) is modulated at the level of the
individual subunit.
The double mutant, R405A/R407A, was inactive and incapable of binding
to the 5'-AMP column through its NAD binding site. Presumably, the
protein was not globally denatured because it appeared to be relatively
stable after synthesis. Interestingly, the double mutant with
asparagine replacing alanine (R405N/R407N) restored structural
integrity while basically reflecting the properties of the individual
alanine mutants, R405A and R407A. The asparagine side chains may have
restored hydrogen bonding capability although they are considerably
shorter than the native arginyl side chains. On the other hand, the
increased bulk and polarity of the asparaginyl side chains over that of
alanine may have provided critical stability in the polypeptide folding
process perhaps by providing alternative interactions. Nonetheless,
even though catalytic activity was mostly restored, cooperativity was
still significantly decreased.
Mutation of Arg-339 to Ala (R339A) produced a protein that bound to the
5'-AMP column but was recovered at such a low level that it was not
possible to do a satisfactory kinetic analysis. This property could not
be overcome by substituting asparagine for alanine (R339N), but could
be reversed to a large extent by lysine (R339K). Because the native
arginyl residue forms hydrogen bonds with main chain carbonyls, the
length of the side chain for hydrogen bonding rather than the presence
of a formal positive charge appears to be critical in this case. The
hydrogen bonding network observed between Arg-339, Tyr-410, Arg-338,
and Glu-387 appears to be critical for serine sensitivity but not so
much for cooperativity or kinetic activity. Mutation of the Arg-338 side chain or complete removal of Tyr-410 significantly decreased the
enzyme's sensitivity to serine. However, a similar result was not seen
when the tyrosine side chain was changed to an alanine. If anything,
the enzyme became more sensitive. This indicates that the tyrosine side
chain itself is not required unless perhaps it plays a small role in
fine tuning the position of Arg-339, but rather the C-terminal carboxyl
at that position is critical.
Removal of the Glu-387 side chain produced little effect, either by
itself or when the C-terminal carboxyl was also missing (compare E387A
to
Y410/E387A and
Y410). These results point to the Arg-338 side
chain as being the critical element. It may possibly serve to stabilize
the orientation of Arg-339 through its effect on the rotational freedom
of the polypeptide chain or by holding the polypeptide chain
transitionally steady at this point.
In general, those mutants that decreased cooperativity are also those
that participate in interdomain interaction within the subunit. Those
mutants that show their greatest effect on sensitivity to serine are
those that participate only in intradomain interactions. What is more
curious is the observation that mutants that presumably decrease
interaction across the domain interface not only decrease cooperativity
but also increase the sensitivity of the enzyme's active site to
inhibition by serine. This suggests a potential separation of pathways
between how the simple act of serine binding results in noncooperative
active site inhibition in the first place and how serine binding also
leads to cooperativity between sites. The question remains as to the
identity of the interactions that govern noncooperative inhibition,
because disrupting the known interactions seems to increase the potency
of serine. The explanation probably lies in the fact that we do not
have a picture of the conformation that occurs in the uninhibited
enzyme because it has not been possible to produce a stable crystal
under these circumstances. Thus, there may be additional interactions
that occur in the absence of effectors that are not present in the inhibited state that account for this situation. Alternatively, these
interface mutations themselves may induce additional interactions at
the interface or in the global orientation of the domains that enhance
the effect of serine. In view of the lack of a crystal structure of the
active enzyme, further probing by specific mutagenesis of this domain
interface will be necessary to address these questions.