From the
Ornithine decarboxylases from Trypanosoma brucei,
mouse, and Leishmania donovani share strict specificity for
three basic amino acids, ornithine, lysine, and arginine. To identify
residues involved in this substrate specificity and/or in the reaction
chemistry, six conserved acidic resides (Asp-88, Glu-94, Asp-233,
Glu-274, Asp-361, and Asp-364) were mutated to alanine in the T.
brucei enzyme. Each mutation causes a substantial loss in enzyme
efficiency. Most notably, mutation of Asp-361 increases the
K
The first committed step in polyamine biosynthesis is the
decarboxylation of ornithine to produce putrescine; this reaction is
catalyzed by a pyridoxal phosphate
(PLP)
We have extended
our previous kinetic analysis
(5) on wild-type mouse,
Trypanosoma brucei, and Leishmania donovani ODC to an
array of potential amino acid substrates. These studies indicate that
enzymes in the eukaryotic ODC family have strict specificity for basic
amino acids and that selectivity between basic amino acids is
accomplished mostly through binding of the substrate in the ground
state. Proteins of known three-dimensional structure with specificity
for positively charged amino acids typically contain an Asp in the
binding pocket which forms a salt bridge with the substrate side chain
(e.g. Asp-189 of trypsin
(6) ; Asp-255 of
carboxypeptidase B (7); Asp-11 of lysine/arginine/ornithine-binding
protein
(8) ). By analogy, the binding pocket of ODC probably
contains an acidic amino acid which interacts with the
The other
possible key roles for an acidic residue are interactions with the PLP
cofactor which support its catalytic reactivity. In aspartate
aminotransferase the protonated N-1 of the pyridoxal ring is stabilized
by a salt bridge to Asp-222
(9) . This interaction is important
to the electron withdrawing capacity of the coenzyme, and thus
facilitates
The eukaryotic ODCs are part of a
family which includes the eukaryotic ODCs, the bacterial
diaminopimelate decarboxylases, eukaryotic arginine decarboxylases, and
prokaryotic arginine decarboxylases related to the Escherichia coli
Spea gene (11, 12). Amino acid sequence information has been
reported for ODC's from 14 species, arginine decarboxylases from
3 species, and diaminopimelate decarboxylases from 6
species
(12) . This wealth of sequence information allows for the
identification of amino acid residues which may be involved in
catalysis or substrate binding by virtue of their conservation
throughout this divergent set of enzymes. Sequence alignment of the 23
related decarboxylases identifies six conserved Asp and Glu residues in
the ODC family, which may be involved in substrate specificity for
basic amino acids or in cofactor reactivity (). We mutated
these residues to Ala in T. brucei ODC. Analysis of these
mutant ODCs, identifies Asp-361 as a substrate binding determinant and
Glu-274 as the residue which interacts with the protonated pyridine
nitrogen of PLP.
The
analysis of binding constants for PLP in the presence of cofactor
analogs, both activators and inhibitors, has been described for other
PLP-dependent enzymes
(17) . The ODC data for the E274A mutant of
T. brucei ODC, approximately 80 data points, was analyzed by
this approach except that a two-substrate model
(18) , which
accounts for the contributions of both E
On-line formulae not verified for accuracy
Mouse ODC catalyzes decarboxylation of all
three substrates more efficiently than the parasite enzymes, as
measured by k
In order to
identify amino acid residues involved in dictating the preference of
ODC for basic amino acids or those involved in the chemistry of
decarboxylation, these six conserved Asp/Glu residues were mutated to
Ala in T. brucei ODC by site-directed mutagenesis. The mutant
proteins were expressed and purified as described under
``Experimental Procedures.'' All six mutant enzymes were
verified to be dimers by gel filtration analysis and to bind
stoichiometric quantities of PLP by spectral analysis (data not shown).
Mutation to Ala at all six positions causes a substantial decrease
in the efficiency of ODC-catalyzed Orn decarboxylation when compared to
the T. brucei wild-type enzyme (I). The
k
These data
demonstrate that Asp-361 is an essential binding determinant. To gain
further insight into the function of Asp-361, the D361E mutant enzyme
was prepared. The K
N-MePLP does not effect wild-type ODC as an inhibitor or
activator (Fig. 1). The binding affinity of PLP for wild-type ODC
was estimated by titration of enzyme activity versus PLP
concentration at a series of substrate concentrations. The apparent
dissociation constant for PLP in the presence of 10 mM Orn, is
about 200 nM for wild-type ODC. V
In the absence of
three-dimensional structural information, Ala-scanning mutagenesis of
evolutionary conserved amino acids can be used to identify structural
determinants of substrate specificity and catalytic reactivity of
enzymes. Six Asp/Glu residues which are conserved in the eukaryotic ODC
family (see ) were mutated to Ala in T. brucei ODC
and the results of kinetic analysis on the purified mutant enzymes are
summarized in the I.
Mutation of Asp-361 to Ala caused
a 2000-fold increase in the K
Conserved acidic residues in ODC also play important roles in
supporting the catalytic reactivity of the PLP cofactor. In aspartate
aminotransferase, Asp-222 forms a salt bridge with the pyridine
nitrogen of PLP
(9, 10) . Mutation to Ala decreases
k
N-MePLP was used as a probe to
identify the Asp or Glu residue which may have a similar role in the
eukaryotic ODC family. Substitution of N-MePLP for PLP
increases k
Mutation of the remaining four conserved acidic residues (Glu-94,
Asp-233, Asp-88, and Asp-364) also had substantial effects on the
efficiency of ODC catalysis. However, at this point we cannot come to
any conclusions about their specific roles. Other possible roles for
conserved acidic residues include the need for a general acid to
protonate the carbanion formed after decarboxylation and the likely
need for additional interactions with the substrate. Of these residues
mutation of Asp-364 caused the most profound changes in enzyme
activity; activity is decreased to a larger extent than for any mutant
ODC which has been described. This includes mutations at Lys-69, the
residue which forms a Schiff base with PLP;
k
The functionally competent form of
eukaryotic ODC is a homodimer with two shared active sites. The
positioning of Lys-69 and Cys-360 on opposite subunits is based on the
observation that activity is restored to 25% of wild-type levels upon
mixing of K69A with C360A
(3, 5, 21) . With the
exception of D361A, all other mutant enzymes are able to restore
activity to the expected levels (e.g. near 25%) upon mixing
with at least some of the other inactive mutants (). This
result demonstrates that the stability of the dimer interfaces have
been unaffected by the mutations, because relative differences in
stability between mutant homodimers in the mixture would decrease the
equilibrium concentration of active heterodimer and result in lower
than expected activity.
D361A appears to be notably different from
the other mutant T. brucei ODCs in its ability to form stable
heterodimers (), suggesting that a nonrandom distribution
of homodimers to heterodimers may be present in the mixtures of D361A
with the other mutant enzymes. Taken together with the proposed role of
Asp-361 in substrate binding, these lower levels of heterodimer
formation may result from the loss of substrate contributions to dimer
stability via the putative salt bridge between Orn and Asp-361. Orn has
been reported to be one of the factors which promotes stable dimer
formation in mouse ODC
(29) . Consistent with this hypothesis,
D361E is able to complement K69A activity to 18% of the wild-type
levels.
Previous studies positioned three residues, Lys-69, Lys-169,
and His-197, in the N-terminal part of the active site
(21) . Our
studies allow the active site map to be extended. The ability of D88A,
E94A, D233A, and E274A to restore activity when mixed with C360A, but
not K69A, demonstrate these residues are contributed to the active site
from the same subunit as Lys-69, while D361A(E) and D364A, which
complement K69A and not C360A, are contributed to the active site from
the same subunit as Cys-360. Thus, the ODC active sites appears to be
formed at the interface between two putative domains, with residues
69-274 present in the N-terminal domain and residues
360-364 participating in the C-terminal domain (Fig. 2).
The amino acid numbering is based on mouse ODC. The sequences of ODC
from mouse, rat, hamster, cow, and human are identical at these
positions and have been listed as mammals, Neurospora crassa and Saccharomyces cerevisiae are included in fungi and
tomato and oat are included in plants. ADC, arginine decarboxylase and
DAPDC, diaminopimelate decarboxylase.
K
The purified mutant enzymes were mixed at 1:1
molar ratio and assayed for activity in the presence of saturating
ornithine (10 mM). Numbers represent the percent of wild-type
activity restored to the system calculated based on the total protein
concentration in the assay. Homodimer activities are displayed as
italics. Theoretically, a 1:2:1 ratio of
homodimer:heterodimer:homodimer should form and activity should be
restored to 25% of wild-type levels, if the two residues are
contributed to the active site from opposite monomers (20).
We thank Dr. Elliott Ross for helpful discussions, and
Mary Quick, Andrea Simmons, and Cory Bentley for technical assistance.
for ornithine by 2000-fold, with little
effect on k
, suggesting that this residue is an
important substrate binding determinant. Mutation of the only strictly
conserved acidic residue, Glu-274, decreases k
50-fold; however, substitution of
N-methylpyridoxal-5`-phosphate for pyridoxal-5`-phosphate as
the cofactor in the reaction restores the k
of
E274A to wild-type levels. These data demonstrate that Glu-274
interacts with the protonated pyridine nitrogen of the cofactor to
enhance the electron withdrawing capability of the ring, analogous to
Asp-222 in aspartate aminotransferase (Onuffer, J. J., and Kirsch, J.
F.(1994) Protein Eng. 7, 413-424). Eukaryotic ornithine
decarboxylase is a homodimer with two shared active sites. Residues 88,
94, 233, and 274 are contributed to each active site from the same
subunit as Lys-69, while residues 361 and 364 are part of the Cys-360
subunit.
(
)
-dependent enzyme, ornithine decarboxylase
(ODC)
(1) . Because polyamines are required for cell growth and
differentiation, ODC has generated interest as a potential drug target
for the treatment of cancer and parasitic infections (e.g. Trypanosomiasis
(2) ). A three-dimensional structure is not
available for any eukaryotic ODC related enzyme, however, biochemical
analysis has led to the identification of several important features of
the enzyme active site. Two identical active sites are formed at the
dimer interface, with Lys-69 contributed to the active site from one
monomer and Cys-360 from the other
(3) . Lys-69 forms a Schiff
base with PLP and Cys-360 was identified as the covalent attachment
site of
-difluoromethyl ornithine
(4) .
-amino
group of Orn. The observation that specificity is achieved in the
ground state suggests that the effects of mutating active site residues
which are involved in substrate recognition might also be largely
confined to changes in K
.
proton extraction. Mutation of Asp-222 to Ala in
aspartate aminotransferase reduced the catalytic efficiency of the
enzyme by 10
, while the K
for
pyridoxamine phosphate was increased by
10
(9, 10) . The loss of enzyme activity upon
mutation of Asp-222 to Ala was partially restored by replacing PLP with
N-methylpyridoxal 5`-phosphate (N-MePLP) as the
cofactor in catalysis
(9) .
Materials
Substrates and the CO detection kit were
purchased from Sigma. Ni
resin was purchased from
Qiagen.
Methods
ODC Expression and Purification
T.
brucei, mouse, and L. donovani wild-type ODC, and the
T. brucei mutant enzymes were expressed in E. coli using the His-TEV vector as described
(5) . ODC
was purified from the soluble fraction of lysed bacterial cells in two
steps: Ni
-agarose column chromatography followed by
Hi-load 16/60 Superdex G-200 gel filtration column chromatography as
described (5). All three enzymes were at least 98% pure after these two
steps. Typical recovery of purified protein from a 6-liter preparation
was 100 mg.
ODC Activity Assay and Kinetic Analysis
Amino acid
decarboxylation was followed spectrophotometrically as
described
(5) . Briefly, CO production is coupled to
NADH oxidation via phosphoenolpyruvate carboxykinase and malate
dehydrogenase. NAD
production is monitored at 340 nm.
The standard assay was done at saturating PLP (20 µM) and
Orn concentrations ranging from 0.05 to 500 mM depending on
the K
of the enzyme being tested. Several
enzyme concentrations were tested for each mutant to ensure that the
reaction was in the linear range. The assays were carried out in a
Beckman DU650 spectrophotometer at 37 °C, pH 8.0. Michaelis-Menten
parameters were calculated using the program k
(Biometallics, Inc).
Protein Determination
The protein concentration in
all ODC samples was determined spectrophotometrically using a
previously determined extinction coefficient of = 0.85
OD(mg/ml)
cm
(5) .
Formation of Mutant Heterodimers
All possible
pairs of the purified T. brucei ODC mutants were mixed
together at 1:1 molar ratios and incubated for 5-60 min before
assay (buffer was either 20 mM Tris 7.5, 2 mM
dithiothreitol, 20 µM pyridoxal phosphate, 0.2% Brij or
assay reagent A). The activity restored in each pair was compared to
the residual activities of both mutants and to the activity of
wild-type ODC equivalent to the total protein concentration in the
assay ().
Gel Filtration Analysis of Mutant T. brucei
ODC
Gel filtration analysis was done using Superose-12 and
Superdex G-200 FPLC columns (Pharmacia) equilibrated in Buffer A (20
mM Hepes, pH 7.5, 50 mM NaCl, 0.5 mM EDTA,
0.015% Brij-35, and 5 mM dithiothreitol). PLP (20
µM) was included for all studies except those involving
N-MePLP. Samples were applied at a protein concentration of
10-20 mg/ml.
Site-directed Mutagenesis
Mutagenesis of T.
brucei ODC was performed by the standard Kunkel technique
(13) in the Bluescript vector (Stratagene) using the M13 helper
phage R408 (Stratagene) and the Kunkel strain BO265. The primers were
as follows, the replaced codon is underlined: D88A,
5`-ACGGGATTTGCCTGCGCAAGCAAC-3`; E94A,
5`-AGCAACACTGCCATACAACGGGTCCGAGGC-3`; D233A,
5`-CACATTCTTGCCATCGGCGGCGGGTTT-3`; E274A,
5`-ATTGTTGCCGCCCCCGGGAGGTAC-3`; D361A, 5`-CCCACATGTGCCGGTCTAGATCAG-3`;
D361E, 5`-CCCACATGTGAGGGGCTCGATCAG-3`; D364A,
5`-ACATGTGATGGGCTCGCTCAGATA-3`. DNA fragments containing the site of
mutation were then subcloned into the expression vector. The structure
of the subcloned fragment was verified by sequence
analysis
(14) .
Synthesis of N-Methyl-pyridoxal
Phosphate
N-MePLP was synthesized and purified as
described
(15) . The structure of the derivatized cofactor was
verified by NMR.
Screening Mutant Enzymes for Activity with
N-MePLP
The initial screening for activity with N-MePLP
was performed using the 1-C-Orn protocol in microtiter
plates
(16) in the presence of PLP or N-MePLP
(50-400 µM). N-MePLP was preincubated with
each enzyme for 2 h prior to assay. Enzyme preparations were partially
depleted of PLP by treatment with 100 mM Cys followed by
exhaustive dialysis as described
(4) .
Estimation of PLP and N-MePLP Binding Affinities to E274A
and Wild-type ODC
Enzyme activity was first measured as a
function of PLP or N-MePLP concentration in the presence of
varying concentrations of Orn (0.05-10 mM for wild-type
and 0.5-50 mM for E274A) to determine the effect of
N-MePLP on the Kfor Orn. To
determine the apparent binding affinities of PLP and N-MePLP
to E274A and the k
for the reaction with
N-MePLP, reaction rates were then collected for multiple
concentrations of PLP and N-MePLP (both cofactors were present
simultaneously) with Orn present at saturating concentration (50
mM). The tested PLP concentrations ranged from 0.1 to 20
µM and the N-MePLP concentrations ranged from 2
to 200 µM. Enzyme concentrations were 20-100
nM for wild-type ODC and 0.15-2 µM for the
mutant enzymes. Holoenzymes were used in the study, free PLP was
removed by gel-filtration on Superdex G-200 equilibrated in buffer A
minus PLP. The final PLP content in these preparations was close to a
1:1 molar ratio with enzyme (determined spectrophotometrically).
PLP and
E
MePLP on the reaction rate, was employed. The apparent
dissociation constants (Michaelis constants) for
PLP(K
) and
N-MePLP(K
), as well
as the maximal velocities for PLP (V
) and
N-MePLP (V
) were obtained by fitting
the data to the equation one using Sigma Plot (Jandel Scientific).
Comparison of Substrate Specificity of Host and
Parasite ODC
The host and two parasite enzymes were tested for
their ability to catalyze the decarboxylation of a number of amino acid
substrates, including several basic amino acids (Orn, Arg, Lys,
2,4-diaminobutyric acid, and diaminopimelate). Detectable activity is
only observed for Orn, Arg, and Lys (). The
k/K
for the
remaining substrates is less than 0.10
M
s
M
, based on the lower limit of
detection of the assay. A previous study of mammalian ODC identified
Lys, but not Arg, as a substrate
(19) . That study was done at
Arg concentrations significantly below K
and this may have prevented detection of the
activity
(19) .
/K
.
The K
for mouse ODC-catalyzed
decarboxylation of Lys is 4-fold less than for T. brucei or
L. donovani ODC (). This result is similar to the
previously identified differences in the K
for Orn between the three species (Ref. 5; ). Mouse
and T. brucei ODC prefer Orn by 250- and 370-fold,
respectively, over Lys and by 1000- and 1400-fold, respectively, over
Arg (). In contrast, L. donovani ODC does not
catalyze Lys decarboxylation as well, Orn is preferred over Lys and Arg
by 1000- and 800-fold (). These substrate preferences are
manifest mostly in K
.
Alanine Scanning of Conserved Asp/Glu Residues of T.
brucei ODC
An amino acid sequence alignment of the 23 eukaryotic
ODC's, arginine decarboxylases, and diaminopimelate
decarboxylases was constructed and will be published
elsewhere.(
)
The sequences of the conserved
acidic residues are displayed in . Of these residues the
only completely invariant one is Glu at position 274. A negative charge
(e.g. an Asp or Glu residue at the position) is conserved
throughout all 23 sequences at positions 88 and 361. A conserved Asp or
Glu is found invariant within the ODCs and within most of the arginine
decarboxylases and diaminopimelate decarboxylases at positions 94 and
233. Asp is found invariant at position 364, except that for the
arginine decarboxylases the position is shifted to 363.
for E94A- and D233A-catalyzed decarboxylation
was decreased by 40- and 130-fold, respectively, while for D88A and
E274A, K
is increased approximately
15-fold for both and k
is decreased
approximately 200- and 50-fold, respectively (I). D364A
had very little residual activity,
k
/K
is reduced by
approximately 10
-fold over the wild-type enzyme. The
mutation of D361A causes a striking 2000-fold increase in
K
, while k
remained relatively unchanged (I).
for D361E-catalyzed
decarboxylation of Orn is substantially higher than for wild-type ODC
(130-fold) but 25-fold lower than the K
observed for D361A. The k
for
D361E-catalyzed Orn decarboxylation is lower than for D361A
(k
was reduced by 15-fold versus wild-type ODC as compared to the 2.5-fold reduction observed for
D361A; I). The weak binding affinity of D361A for Orn
precluded inhibitor analysis. However, D361E could be tested for its
ability to bind the product putrescine as an inhibitor of the reaction.
Putrescine inhibited D361E-catalyzed decarboxylation of Orn with a
K
of 35 ± 3.9 mM, a value
100-fold higher than for wild-type T. brucei ODC
(5) .
N-MePLP as a Substitute for Pyridoxal Phosphate
To
test whether any of the mutant ODCs would be more active in the
presence of N-MePLP, this cofactor analog of PLP was
synthesized and purified as described
(15) . Partially resolved
wild-type and mutant apoenzymes were tested for activity in the
presence N-MePLP as a substitute for PLP in the assay mixture.
Apoenzyme preparations were obtained by partial depletion of PLP by
treatment with Cys as described
(4) . Only about 40% of the PLP
could be removed from the wild-type ODC by this method and some
aggregation and enzyme inactivation was observed. Of the enzymes
tested, N-MePLP affected only E274A. The catalytic efficiency
of E274A was significantly enhanced by the substitution of
N-MePLP for PLP in the reaction (Fig. 1). In contrast,
N-MePLP was inactive as a cofactor in the reactions catalyzed
by wild-type T. brucei enzyme or for the mutant ODCs, D88A,
E94A, D233A, D361A, D364A, K69A, and C360A even in the presence of up
to 400 µMN-MePLP.
Figure 1:
Complementation of E274A activity with
N-methylpyridoxal phosphate. The dependence of
k on PLP (
,
) or N-MePLP
(
,
) concentration is plotted for wild-type T. brucei ODC (open symbols) and E274A (closed symbols).
The kinetic constants were calculated for a range of Orn concentrations
(1-50 mM) in the presence of PLP (0.1-20
µM) or N-MePLP (2-200 µM). The
PLP concentrations for the assays in the presence of N-MePLP
are equal to the enzyme concentration, 0.02 µM for
wild-type ODC and 0.3 µM for
E274A.
A more detailed kinetic
analysis of E274A with N-MePLP as a cofactor was performed.
Because of the difficulties in preparing apoenzyme, holoenzymes
containing equimolar amounts of bound PLP were used for these studies.
Kinetic data were collected for multiple Orn concentrations at
different fixed N-MePLP concentrations (Fig. 1). The
reaction rate was increased substantially in the presence of
N-MePLP, while Kfor Orn was
identical to that observed with PLP as the cofactor. The
k
for Orn decarboxylation by N-MePLP
substituted E274A could only be approximated by this simple analysis
due to the presence of PLP in all enzyme samples. To overcome this
problem, data were collected at multiple concentrations of
N-MePLP and PLP at saturating concentrations of Orn (50
mM). The data were fit to a two-substrate model (see
``Experimental Procedures''). The k
for Orn decarboxylation by E274A in the presence of PLP obtained
by this two-substrate model was identical to that calculated
independently in the absence of N-MePLP (). The
apparent dissociation constant
(K
) for PLP and an apparent
dissociation constant (K
) for
N-MePLP were calculated to be 1.8 ± 0.2 and 23 ± 3.2 µM, respectively. The low activity of E274A is
enhanced to near wild-type levels by the substitution of
N-MePLP for PLP in the assay buffer (Fig. 1);
k
is increased 30-fold to 6 s
at saturating concentrations of N-MePLP.
increased from 30 to 60% of the level measured in the presence of
saturating PLP, as the concentration was increased from 150 to 300
nM (Fig. 1).
Complementation Analysis of Asp/Glu to Ala Mutant
Enzymes
The conclusion that ODC has shared active sites is based
on the observation that activity is restored to 25% of wild-type levels
upon co-expression
(3) or mixing of the inactive K69A and C360A
mutant enzymes
(5, 21) . We have previously demonstrated
that this experimental approach is valid for T. brucei ODC
(5) . The mutant Asp/Glu to Ala T. brucei enzymes were tested for their ability to restore activity when
mixed with the inactive K69A or C360A ODC. This study allows us to
evaluate their ability to form functional dimers and to localize the
corresponding residues in the shared active site with respect to Lys-69
and Cys-360. Activity is restored to 25% of wild-type levels when D88A,
E94A, D233A, or E274A are mixed with C360A but not if they are mixed
with K69A or with each other (). D364A is able to
complement K69A, D88A, E94A, D233A, and E274A, restoring activity to
levels of 5-20%, but is unable to complement C360A
(). D361A, poorly, but consistently complements the
mutants at positions 69 through 274 to levels of 3-8%, but does
not complement C360A or D364 at all. However, the other mutant in this
position, D361E, is able to complement up to 18% of wild-type activity
when mixed with K69A.
DISCUSSION
Mouse, T. brucei, and L. donovani ODC
exhibit strict specificity for basic amino acids as substrates. A
comparison of the specificity constant
(k/K
) reveals that
all three enzymes also exhibit a strong preference for Orn over Lys and
Arg (). We had previously demonstrated that for
ODC-catalyzed Orn decarboxylation K
is
likely to be a true dissociation constant
(5) . Therefore, the
50-500-fold increase in K
for Lys
or Arg over Orn suggests that ODC discriminates between the basic amino
acids in the ground state. Very little additional specificity is gained
in the transition state, as k
only contributes
2-7-fold of the observed effect to the specificity constant
(k
/K
). The
exception is the 25-fold higher k
observed for
L. donovani ODC-catalyzed decarboxylation of Orn over Lys. The
finding that a 10
-fold substrate preference for Orn over
Arg in ODC is achieved mostly in K
is
remarkable. For many enzymes, substrate discrimination is reflected
through interactions which effect k
, thus
allowing the binding energy of the correct substrate to be used to
accelerate the rate of catalysis (e.g. the 10
-fold
preference of trypsin for Arg over Phe
(22) ; the
10
-fold preference of lactate dehydrogenase
(23) for
pyruvate over oxaloacetate; and the 10
-fold preference of
papain for Phe over Arg
(24) ).
for Orn
decarboxylation, without significantly lowering
k
, thus identifying Asp-361 as an essential
substrate binding determinant. The binding affinity of D361E for
putrescine is reduced to the same extent as for Orn
(K
= K
= 30 mM) suggesting that as for wild-type ODC,
the K
is a reflection of the true
dissociation constant. Therefore, in the D361A or D361E mutant enzymes,
the observed increases in K
most likely
reflect a loss in binding affinity for the ground state substrate
structure similar to the observations with alternate substrates. These
data suggest that the
-carboxylate of this residue interacts
directly with the substrate via formation of a salt bridge with either
of the two amino groups of the substrate. The magnitude of the effect
(
G of 5 kcal/mol; I) is in keeping with
the loss of a hydrogen bond between two charged residues, based on
similar studies in other systems (e.g. trypsin
(25) ,
carboxypeptidase
(26) ,
-lactamase (27), and lactate
dehydrogenase
(28) ). The observation that mutation of Asp-361 to
a larger group that preserves the charge (D361E) resulted in a
K
for Orn that is lower than D361A, but
still 130-fold higher than wild-type, also supports this
interpretation. Alternatively, mutation of D361A may have caused a
conformational change in the enzyme, such as a domain or subunit
rotation, resulting in an enzyme with poor substrate affinity.
/K
by greater
than 4 orders of magnitude, however, activity is increased
4-20-fold by substitution of N-MePLP, which has a fixed
positive charge on the pyridine nitrogen, for PLP in the
reaction
(9) .
for E274A-catalyzed Orn
decarboxylation 30-fold, bringing it to within 75% of the wild-type
k
; in contrast, there was no effect on any of
the other Asp or Glu mutant enzymes or on wild-type ODC. The finding
that N-MePLP does not bind to wild-type ODC even at
concentrations up to 400 µM, while it binds tightly to
E274A (K
= 23
µM), suggests that a cavity in the enzyme active site,
which can accommodate the additional methyl group on the pyridine
nitrogen, has been created by the replacement of Glu-274 with the
smaller Ala residue. Additionally, the apparent binding constant of PLP
for E274A is at least 20-fold higher than for wild-type ODC. Cofactor
binding affinity was also reduced in the D222A mutant of aspartate
aminotransferase
(10) . Thus, analogous to Asp-222 in aspartate
aminotransferase, we propose that Glu-274 forms a salt bridge to the
protonated pyridine nitrogen of PLP in ODC and thereby functions to
stabilize the carbanion generated by decarboxylation of Orn.
Interestingly, N-MePLP is a more potent activator of E274A ODC
than was observed for the D222A mutant of aspartate aminotransferase.
The fact that in the presence of N-MePLP, k
for E274A-catalyzed decarboxylation is restored to near wild-type
levels, suggests that the sole role of Glu-274 in catalysis is
maintenance of the positive charge on the pyridine nitrogen of PLP.
/K
is decreased
by only 700-fold for K69A
(4) , while it is decreased by greater
than 10
-fold for D364A, pointing to an essential role for
Asp-364 in ODC catalysis.
Figure 2:
Schematic representation of the putative
eukaryotic ODC active site. Amino acid residues potentially
contributing to the shared active site of ODC, as determined previously
(21) and in this study by mutagenesis, are shown with respect to the
likely N-terminal and C-terminal domains. Two residues with established
function, Lys-69 (4) and Glu-274, are outlined. The PLP cofactor is
shown as a Schiff base with Orn.
Thus far, all identified residues which form interactions with PLP
(Lys-69 and Glu-274) are found in the N-terminal domain. Our previous
characterization of mouse/T. brucei cross-species heterodimers
suggests that the -carboxylate of Orn interacts with the Lys-69
side of the active site
(5) ; however, the remaining contacts to
substrate which have been identified appear to be in the C-terminal
domain. This includes, Cys-360, the site of modification by
-difluoromethyl ornithine
(4) and the likely interaction of
Asp-361 with one of the amino groups in the substrate.
Table:
Conserved acidic residues in the Eukaryotic ODCs
Table:
Substrate specificity of host and parasite ODCs
, mM;
k
, s
;
k
/K
is in
M
s
and specificity is
the ratio of k
/K
for Orn to the other substrate. Data was collected at saturating
concentrations of PLP (20 µM). The results for Orn were
taken from Ref. 5. Kinetic constants were calculated by Lineweaver-Burk
analysis using the program k
.
Table: 1828742767p4in
ND, not
determined.(119)
Table:
Formation
of mutant heterodimers
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.