The Direct Measurement of Thermodynamic Parameters
of Reactive Transient Intermediates of the
L-Glutamate Dehydrogenase Reaction*
Steven J.
Maniscalco,
Jon F.
Tally,
S.
Welsh
Harris, and
Harvey F.
Fisher
From the Laboratory of Molecular Biochemistry, Veteran Affairs
Medical Center and the Department of Biochemistry, University of Kansas
Medical Center, Kansas City, Missouri 64128
Received for publication, January 21, 2003, and in revised form, February 10, 2003
 |
ABSTRACT |
In a previous report (Fisher, H. F.,
Maniscalco, S. J., and Tally, J. (2002) Biochem. Biophys. Res.
Commun. 287, 343-347) we demonstrated the capability of the
"Le Chatelier forcing method" of producing stable solutions
containing substantial amounts of transitory enzyme intermediate
complexes that can otherwise be observed only fleetingly in the
millisecond time range. The method requires nothing more than
running an enzyme reaction using forcing concentrations of reactants
against an equally forcing concentration of products until equilibrium
is attained. Here we have applied this approach to the measurement of
the thermodynamics of several such reactive (and normally transient)
intermediate complexes of the bovine liver
L-glutamate dehydrogenase-catalyzed reaction. At pH
9.5 and 20 °C, we observe both the enzyme-NADPH-
-iminoglutarate and enzyme-NADPH-
-carbinolamine complexes at concentrations whose sum accounts for 70% of the total enzyme. The pH dependence of these
two complexes under equilibrium conditions provides thermodynamic parameters for both the protonated and the unprotonated forms of each
of these two entities as well as those of the
enzyme-NADP-L-glutamate complex. The equilibrium
concentrations of each of these reactive complexes are compared with
their corresponding transient steady-state values.
 |
INTRODUCTION |
Establishing structure-function relationships is a focal point in
the determination of mechanisms of enzymatic catalysis. The measurement
of thermodynamic differences between related enzyme complexes is an
important step in such endeavors. Such measurements have been
necessarily limited to non-reactive complexes; the structures of the
mechanistically and energetically vital reactive intermediate complexes
have been typically derived solely on the basis of chemical intuition.
Two recent experimental developments now provide the basis for the
direct measurement of differences in thermodynamic parameters of
spectroscopically identified reactive intermediate complexes whose
kinetic competence has been verified. These developments are 1) the
transient-state multiwavelength spectroscopic approach, which can
produce resolved component reaction time courses of enzyme reactions
(1, 2); and 2) the Le Chatelier forced equilibrium approach,
which produces significant concentrations of reactive transient
intermediate complexes in solution under true thermodynamic equilibrium
conditions (3, 4). Here, we report the results of the first set
of such thermodynamic measurements on reactive intermediates of the
bovine liver L-glutamate dehydrogenase (blGDH)1 reaction.
The Experimental System
The stoichiometry of the glutamate
dehydrogenase reaction is shown in Equation 1. The capital letter immediately below each reactant or product species is used to designate
that entity in the various enzyme (E) complexes to be discussed.
|
(Eq. 1)
|
The generally accepted mechanism of the amino acid
dehydrogenase-catalyzed oxidative deamination reactions is shown in
Scheme 1, which portrays the chemical
state of the
-carbon atom of the substrate in the sequence of
central complexes.
In the blGDH-catalyzed
reaction2 we have shown that
the nicotinamide moiety of the coenzyme has the following
spectroscopic properties relative to those of the 340-nm peak of
free NADPH:HEOG is colorless, EOG' is a
weakly absorbing, red-shifted highly fluorescent charge transfer
complex (5), ERI (enzyme-NADPH-
-iminoglutarate) is highly
blue-shifted, ERC (enzyme-NADPH-
-carbinolamine) is highly
red-shifted (1), and ERK (enzyme-NADPH-
-ketoglutarate) is
slightly blue-shifted.
The Le Chatelier Force (Ammonia Dam) Approach to the Study of
Reactive Central Complexes
Here, we allow an amino acid
dehydrogenase reaction containing high concentrations of the enzyme,
the amino acid substrate, NAD(P), and a product, ammonia, to reach the
equilibrium shown in Scheme 2, a process
which is usually complete in 3-40 min.
This application of high concentrations of free reactants from both
ends of the chemical reaction tends to drive the reaction toward the
center, building up high and readily observable concentrations of the
enzyme central complexes.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Beef liver glutamate dehydrogenase obtained from
Sigma as an ammonium sulfate suspension was dialyzed against three
changes of 0.1 M potassium phosphate, 0.05 M
MES, and 0.05 M CHES buffer at the required pH and was
filtered through Norit A and a 0.45-µm filter. The pH was
adjusted with potassium hydroxide and phosphoric acid. The
concentration of the enzyme was measured spectrophotometrically at 280 nm,
= 54,400 M
1 cm
1.
Potassium phosphate, potassium hydroxide, phosphoric acid, MES, CHES,
NADP, and ammonium sulfate were purchased from Sigma.
L-glutamic acid was purchased from Calbiochem. All were
used without further purification.
Methods--
Spectra were collected in conventional 1-cm quartz
cuvettes in a Hewlett-Packard diode-array model 8450A spectrophotometer equipped with magnetic stirring and thermostatted by pelletier devices
to provide temperatures accurate to within 0.1 °C. The initial
concentrations were [E] = 5-40 µM; [O] = 380 µM; [G] = 45 mM; [N] = 300 mM, in a 0.1 M potassium phosphate, 0.05 M MES and 0.05 M CHES buffer adjusted to the
appropriate pH. The reference cuvette contained corresponding
concentrations of G, O, and N in the same buffer. A spectrum of
E alone at the same concentration as in the experimental
solution was also subtracted from that of the experimental solution.
Except where otherwise stated, the results reported here were obtained
at 20 °C. Each spectrum was resolved into the sum of
species-relatable spectroscopic components as previously described (1).
Possible contributions were considered from the following array of
model spectra: free NADPH (R), the enzyme-NADPH complex
(ER), the enzyme-NADPH-L-glutamate complex (ERG), and the ERK complex obtained from stable
complexes as well as ERI and ERC complexes
obtained from the resolution of transient-state absorbance
versus time and wavelength arrays. Application of Cramer's rule to a determinant made up of Beer's law equations using these model spectra and the experimentally observed one permitted a least
squares regression solution using Matlab 6 software (1). The filtration
experiment was carried out by loading an equilibrated solution into a
10-ml syringe equipped with a Swinney adaptor holding a 0.22-µm
Millipore filter and expressing various amounts of filtrate.
 |
RESULTS |
A typical resolution of a spectrum of a forced equilibrium
solution into distinct components is shown in Fig.
1.3 It can be
seen that the only R-containing complexes
present at measurable concentrations are the highly blue-shifted
ERI complex and the highly red-shifted ERC
complex. Given the reaction conditions, the presence of free
E is precluded, and the remainder of the enzyme that is
unaccounted for must be a mixture of HEOG and
EOG' forms. This qualitative pattern of complexes is
observed throughout the observed pH range. The results of a series of
such resolved spectra over the pH range of 6.0-9.5 are shown by
the data points in Fig. 2.

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Fig. 1.
Resolution of the absorption spectrum of a
blGDH Le Chatelier forced equilibrated reaction solution. The
reaction conditions are pH 7.6, 20 °C, [ET] = 22 µM. Other conditions were as described under
"Methods." The open circles labeled
A(Total) represent the experimental absorbance
spectrum. The solid lines labeled A(R),
A(ERC), and A(ERI) represent the component
contributions, and their sum is represented by the solid
line through the open circles. The relative concentration of the
HERI complex is too small throughout the pH range to appear on this
scale. The residuals of the fit of this sum to the experimental data
are shown in the lower panel.
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Fig. 2.
The pH dependence of resolved reactive
intermediate complexes. The open triangles ( ) show the relative
concentrations of the ERI complex. Each point represents the
average of 3-20 experimental values. The open squares ( ) represent
the relative concentrations of the ERC complex. All
concentrations are expressed as fractions of the total enzyme
[ET]. The solid lines represent the
best overall fit of the data to Equation 1. The vertical
lines through each symbol show the estimated probable
error. [ET] varied from 5-40
µM.
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Evidence that the Le Chatelier forced systems we have used in this work
are at (or at least very close to) true thermodynamic equilibrium is
provided by the following experimental observations: 1)
The equilibrium constant for the O +
R +
+ K +
can be expressed as Keq = [R]2
[
]
[
+]/(([O]
[RT])
[
]) for a reaction started with O, G, and N and an
amount of enzyme too low to permit the absorption of enzyme complexes
to contribute to the observed signal. The components indicated by the
superbar are considered to be "buffered"; that is, their
concentrations remain constant throughout the reaction. The value of
the equilibrium constant was measured over a range of very low enzyme
concentrations. The observed pH-dependent value of 5.3 × 10
14 M2 obtained at 20 °C
agrees very well with that of 7.3 × 10
14
M2 reported by Subramanian (6) for the same
reaction run at 25 °C, corrected for the
H0 of 17 kcal/mol
1 reported in the same study. Fig.
3 shows the values of
G0
calculated from Keq values from the same
reaction at high enzyme concentration and varying pH using values of
[R]free obtained from the spectral component analysis.
The variations from the theoretical value (indicated by the arrow in
the figure) are not sufficiently large enough to effect our conclusions
materially. 2) Addition of small amounts of
-ketoglutarate to a
presumably equilibrated reaction mixture rapidly reverses the reaction.
3) Forcing an equilibrated solution through a 0.22-µm filter in a syringe fitted with a Swinney adaptor produced a filtrate containing the essentially same concentration of free NADPH as that calculated by
spectroscopic resolution in the original equilibrated solution. 4)
Raising or lowering the temperature of a high enzyme equilibrated reaction mixture substantially and reversibly changes both the concentration of free R in accordance with the positive
H0 of the chemical reaction and alters the relative
concentrations of the ERI and ERC components
(data not shown). The reversal of temperature changes from 0 to
35 °C showed only a small hysteresis effect.

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Fig. 3.
The pH dependence of
G . The solid
horizontal line is the arithmetic average of all the
points. The dashed horizontal lines show the probable
error limits of each point. The arrow designates the value
of Keq calculated from Ref. 5.
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Given the observations and assumptions described above, the system can
now be described by Scheme 3, which can
be evaluated by Equations 2-7.
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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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(Eq. 6)
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(Eq. 7)
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The values of each of these pH-independent equilibrium constants
were obtained from a simultaneous fit of the data to the whole set of
Equations 2-7. The resulting values, along with those of the standard
free energies (calculated from
G
are provided in Table
I. The solid lines in Fig. 2 represent
the fit with these parameters to the pH dependence of the
concentrations of ERItotal and
ERCtotal. Fig. 4
shows the concentrations of the individual ionic species as a function
of pH. In Fig. 5 we show the
differences in
G0 for the various species, assuming
pH = 7.6 and arbitrarily setting
G
= O.

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Fig. 4.
The pH dependence of equilibrium
concentrations of ionic species of reactive intermediates. The
curves are calculated by application of Equations 2-7 to the data
shown in Fig. 2. The relative concentration of the HERI complex is too
small throughout the pH range to appear on this scale.
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Fig. 5.
Free energies ( G) of
the various enzyme complexes denoted in Scheme 3. All
values are calculated relative to that of the HEOG complex,
assigning a pH = 7.6. Solid bars refer to protonated
forms, and open bars refer to their unprotonated
counterparts.
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 |
DISCUSSION |
Observed Phenomena--
The results shown in Fig. 5 lead
to the potentially important conclusion that both HERI and
ERI are thermodynamically highly unfavored structures,
whereas the unprotonated ERC structure is only slightly less
stable than the HEOG and EOG complexes. We now
interpret the data of Fig. 2 in the context of Scheme 3. The data show
that at pH values at or above 8, 70% of the total enzyme is tied up as
ERI or ERC complexes under the conditions of the experiment and that throughout the pH range, the concentration of the
carbinolamine complex exceeds that of the
-imino complex by at least
6-fold. It is also apparent from Fig. 2 that the pH dependence of both
the reactive intermediate complexes observed in this system involves
the ionization of a single proton. Because the apparent pK
values of these complexes differ, it is conceivable that we could be
observing the ionization of a different functional group in each case.
However, evidence from other studies argues strongly that, to the
contrary, we are titrating the same functional group in all three
complexes and that the pKapp differences reflect differences in the local interactions of that group with the varying chemical bond makeup of each individual complex. Piszkewicz et al. (6) showed that blGDH could be inactivated by pyridoxal or
pyridoxal phosphate and that at pH values of less than 10 only one
molecule of either reacted rapidly. They also showed that incorporation
of the pyridoxyl moiety into the protein involved imine formation with
(and only with) lysine 126 of the enzyme. They found an apparent
pK = 8.0 for the reaction. Subsequent studies have
found this particular residue to be conserved in all the pyridine
nucleotide-linked
-amino acid dehydrogenases (7), and its unmodified
presence is an obligatory requirement for catalysis.
Assuming then that the marked differences in the pK values
observed represent only shifts in the pK of the same
residue, it follows that they must be because of corresponding
differences in the bonding state of that residue in each of the three
different complexes. The reaction shown in Equation 8 results in
Equations 9 and 10.
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(Eq. 8)
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(Eq. 9)
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(Eq. 10)
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The feature shown in Equation 8 occurs repeatedly throughout
Scheme 3 on which we have based our interpretation of the observed pH
dependences of the EOG, ERI, and ERC
complexes and would appear to provide a sound basis for explaining our
results. It may also be noted that the apparent pK values
defined by the inflection points of the ERI and
ERC pH dependence curves shown in Fig. 2 do not correspond
to any single pK value obtained from our analysis. Thus,
such a phenomenological pK value has no real physical
significance and represents only the result of the assembly of
relationships of the type shown in Equation 8, which occur in a system
described by the model shown in Scheme 3.
A comparison of both the absolute and relative concentrations of the
ERI and ERC complexes at equilibrium, shown as
dashed horizontal lines, and those in the transient local steady state, shown as solid curved lines in Fig. 6,
reveals some striking differences between the compositions of the
thermodynamic and kinetic states. At equilibrium, the concentration of
ERC is far greater than that of ERI at all pH
values. In the transient state, on the other hand, ERI
dominates the early burst phase and remains at higher concentrations
than ERC throughout the major portion of the reaction time
course. The figure shows that at pH 7.6 the transient-state concentration of EOGT is about twice that
of its equilibrium concentration. This situation may be expressed in
any of three equivalent sets of conceptual terms. 1) In terms of
classic chemical kinetics, EOG exhibits some degree of
kinetic rather than purely thermodynamic control. 2) In the language of
absolute reaction rate (or activated complex) theory, EOG
formation is preceded by a low transition state barrier and followed by
a relatively high barrier. 3) In steady-state kinetic terms,
EOG itself has a low forward commitment factor and must be
preceded by a complex having a relatively high commitment factor.

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Fig. 6.
A comparison of transient-state and
equilibrium values of components under Le Chatelier forced
conditions. The solid lines represent the
transient-state kinetic time courses of the resolved reaction time
courses carried out at 20 °C, pH 7.6, in the presence of 0.3 M ammonia. The horizontal dashed lines
designate the values of each resolved component at equilibrium.
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The transient local steady-state concentration of the ERI
complex is nearly identical to its equilibrium value. Therefore, ERI is 1) under thermodynamic control, 2) lies between low
transition barriers, and 3) its forward commitment factor is
effectively balanced by those of the preceding and following complexes.
The transient local steady-state concentration of the ERC
complex is 10-fold lower than its equilibrium value. Therefore, 1) its
concentration is dominated by kinetic control, 2) the transition state
barrier preceding its formation is higher than that following it, and
3) its forward commitment factor is substantially greater than that of
the preceding complex.
It can be seen from Fig. 5 that the unprotonated forms of both the
imino and carbinolamine complexes are thermodynamically more stable
than their corresponding protonated forms at pH
7.6. Although
high reactivity of minor species is always a possibility, the
predominance of unprotonated post-hydride transfer species fits in well
with the postulated kinetic mechanism (9).
Non-observed Phenomena--
The lack of appearance of any of the
relatively tight blue-shifted ERK complex from the
equilibrium mixture is easily explained by the driving of the
ERK + N
ERI far to the
ERI form because of the presence of the high concentration
of ammonium. The absence of any trace of the red-shifted ERG
complex is somewhat more difficult to account for. This dead-end
inhibitory complex, although slow to form, is so very tight that it
constitutes the final complex in the transient-state reaction, and the
release of free NADPH from the ERG complex is the
rate-limiting step in the steady-state forward reaction. Although the
inhibition of the GDH reaction by ammonia is quite complex, we will
show elsewhere that the high concentration of ammonia is successful in
competing with the
-amino group of L-glutamate at
the ammonia binding site of the enzyme, forcing the substrate to bind
in a mode in which hydride transfer is
precluded.4
In a previous report on the transient-state resolved component time
courses of the Clostridium symbiosum glutamate dehydrogenase (csGDH) reaction, we have demonstrated the occurrence of isomeric forms
of ERI, ERC, and ERK complexes (3).
These isomeric forms differ from the previously identified entities in
that their spectral 340-nm peak is unshifted and therefore
spectroscopically indistinguishable from that of free NADH. Their
existence has been established and their location on the reaction
coordinate has been determined by the kinetic competence of this
anomalous free R to participate actively in the reaction at several
mechanistically separate points. We have ascribed the characteristic of
an unshifted spectra to open forms of the enzyme complexes in which the
reduced nicotinamide moiety is exposed to a largely aqueous
environment, in contrast to the hydrophobic environment of the closed
forms. "pH jump" experiments performed on the csGDH transient-state
reaction confirmed an obligatory alternation between open and closed
forms along the reaction pathway (11). Had such open unshifted
complexes been present in significant concentrations in the
equilibrated blGDH solutions we have described here, they would
necessarily have led to anomalously high values for the stoichiometric
equilibrium constant Keq. No such effect was
observed in any of our blGDH experiments. Furthermore, pH jump
experiments on blGDH transient-state studies reported elsewhere (4)
showed the occurrence of measurable amounts of open forms only in the
prehydride transfer phase of the reaction. The lack of appearance of
open forms in the equilibrated blGDH solutions reported here suggests
that the basis of this aspect of the difference in kinetic behavior of
the two GDH species is thermodynamic in nature; the "open
closed" equilibria lie much farther to the right in
the blGDH reaction.
We have recently shown the presence of a generally conserved active
site motif in this class of enzymes (12). It consists of a close-packed
atomic tetrad made up of the
-amino nitrogen atom of lysine 126, an
oxygen atom of the carboxylate group of aspartate
168,5 the nitrogen atom of
the
-amino group of the substrate, and the oxygen atom of a water
molecule hydrogen bonded to lysine 126. Superposition of x-ray crystal
structures of different complexes of a variety of these dehydrogenases
showed that these tetrads very nearly, but not quite exactly, mapped
each other. Although the atom-to-atom center distances around the
tetrad averaged about 2.8 Å, they differ individually by as much as 1 Å. Computer modeling of these structures correspondingly predicted a
wide variety of hydrogen bonding patterns in this motif. Fig. 5
indicates
G0 differences between the EOG,
ERI, and ERC structures of the order of 1-5
kcal/mol
1. This is about the range of values expected for
either the formation of a strong H-bond or the difference between the
breaking of one weak bond and the formation of a different strong
H-bond. Although still a matter of conjecture, this agreement may not
be coincidental.6
Finally we note that although the thermodynamic characterization of the
transient ERI and ERC complexes provided here
presents an essential but very elementary step in the full
understanding of the structural and chemical nature of these
catalytically important entities, the approach whose application we
have demonstrated may contribute certain features that could be quite
useful in extending studies of enzymatic catalysis to a deeper level of understanding. At the present time, the methods available for determining the structures of enzymes in various complexes (such as
x-ray crystallography, neutron diffraction, NMR, IR) all require substantial periods of time for their completion. Their application therefore has been restricted to unreactive complexes. On the other
hand, as seen in Fig. 1, the ERC and ERI
complexes remain intact for at least one month. Enzyme assays conducted
on aliquots of the equilibrated solution show no loss or enzyme
activity during that time period. Thus, the Le Chatelier forced
equilibrium approach permits the application of such time-requiring
methods to the examination of the transient reactive intermediate
complexes that constitute the heart of an enzymatic reaction.
 |
FOOTNOTES |
*
This work was supported in part by the Department of
Veterans Affairs and by Grant MCB-9513398 from the National Science
Foundation.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: Research Service,
Veterans Affairs Medical Center, 4801 Linwood Blvd., Kansas City, MO
64128. Tel.: 816-861-4700 (ext. 7156); Fax: 816-861-1110; E-mail:
hfisher@kumc.edu.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M300692200
2
Subsequent to publication of our paper (3) in
which we introduced what we presumed to be a novel method and during
the process of review of this paper, it came to our attention that the
approach had in fact already been utilized successfully and elegantly
in a paper from the laboratories of K. Johnson and K. Anderson in which
they applied 13C NMR to a forced equilibrium system to
identify and characterize an enzyme-bound tetrahedral
intermediate in the 5-enolpyruvoylshikimate 3-phosphate synthase
reaction (4).
3
As a refinement to the spectroscopic resolution
procedure described in Ref. 1, we now correct the reaction spectrum for the contribution of the small loss of NADP to form free R and the ERI
and ERC complexes.
4
S. K. Saha, J. F. Tally, S. J. Maniscalco,
S. A. Adediran, and H. F. Fisher, unpublished data.
5
Although residue 168 was assigned as an Asn in
the blGDH crystal structure reported by Peterson and Smith (13), it is
in fact a highly conserved Asp residue in all other GDH structures. Dr.
T. Smith has informed us that the Asn assignment was based on very
early amino acid sequence studies (7) and that residue 168 is most
probably an Asp.
6
A detailed description of the relationships
between the geometric pattern of active site residues and the
corresponding catalytic and thermodynamic properties in the various
complexes identified here will be presented elsewhere.
 |
ABBREVIATIONS |
The abbreviations used are:
blGDH, bovine
liver GDH;
GDH, glutamate dehydrogenase;
E, enzyme;
O, oxidized coenzyme;
I, iminoglutarate;
R, reduced coenzyme;
G, L-glutamate;
C, carbinolamine;
EOGT, the sum of
protonated and unprotonated EOG;
K,
-ketoglutarate;
EOG, enzyme-oxidized coenzyme-L-glutamate complex;
EOG', isomerized EOG;
HEOG, the protonated form
of EOG;
ER, the enzyme-NADPH complex;
ERG, ER-L-glutamate;
ERK, ER-
-ketoglutarate;
ERC, ER-
-carbinolamine;
ERI, ER-
-iminoglutarate;
MES, 2-(N-morpholino)ethanesulfonic acid;
CHES, 2-(cyclohexylamino)ethanesulfonic acid, 5-enolpyruvoyl shikimate
3-phosphate.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.