©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Acidic Residues Important for Substrate Binding and Cofactor Reactivity in Eukaryotic Ornithine Decarboxylase Identified by Alanine Scanning Mutagenesis (*)

Andrei L. Osterman , Lisa N. Kinch , Nick V. Grishin , Margaret A. Phillips (§)

From the (1) Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 Kfor 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.


INTRODUCTION

The first committed step in polyamine biosynthesis is the decarboxylation of ornithine to produce putrescine; this reaction is catalyzed by a pyridoxal phosphate (PLP)() -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) .

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 -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.

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 proton extraction. Mutation of Asp-222 to Ala in aspartate aminotransferase reduced the catalytic efficiency of the enzyme by 10, while the Kfor 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) .

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.


EXPERIMENTAL PROCEDURES

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 Kof 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).

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 EPLP and EMePLP 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).

On-line formulae not verified for accuracy


RESULTS

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/Kfor the remaining substrates is less than 0.10M sM, 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 Kand this may have prevented detection of the activity (19) .

Mouse ODC catalyzes decarboxylation of all three substrates more efficiently than the parasite enzymes, as measured by k/K. The Kfor 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 Kfor 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.

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 for E94A- and D233A-catalyzed decarboxylation was decreased by 40- and 130-fold, respectively, while for D88A and E274A, Kis increased approximately 15-fold for both and k is decreased approximately 200- and 50-fold, respectively (I). D364A had very little residual activity, k/Kis 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).

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 Kfor D361E-catalyzed decarboxylation of Orn is substantially higher than for wild-type ODC (130-fold) but 25-fold lower than the Kobserved 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 Kof 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.

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 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 Kis likely to be a true dissociation constant (5) . Therefore, the 50-500-fold increase in Kfor 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 Kis 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) ).

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 Kfor 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 Kis a reflection of the true dissociation constant. Therefore, in the D361A or D361E mutant enzymes, the observed increases in Kmost 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 Kfor 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.

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/Kby 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) .

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 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.

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/Kis 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.

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).


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

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.


  
Table: Substrate specificity of host and parasite ODCs

K, mM; k, s; k/Kis in M s and specificity is the ratio of k/Kfor 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

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).



FOOTNOTES

*
This work was supported by Welch Foundation Grant I-1257, American Heart Association, Texas affiliate, Grant 93G-08S, and National Institutes of Health Grant R01 AI34432-01A2 (to M. A. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 214-648-3637; Fax: 214-648-2971.

The abbreviations used are: PLP, pyridoxal 5`-phosphate; ODC, ornithine decarboxylase; N-MePLP, N-methylpyridoxal 5`-phosphate; mutant T. brucei ODCs are referred to by their single letter amino acid codes, e.g. the Asp-88 to Ala mutant enzyme is referred to as D88A.

N. Grishin, M. A. Phillips, and E. Goldsmith, manuscript submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Elliott Ross for helpful discussions, and Mary Quick, Andrea Simmons, and Cory Bentley for technical assistance.


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