Stereochemistry and Function of Oxaloacetate Keto-enol Tautomerase*

Oxaloacetate keto-enol tautomerase, partially purified from porcine kidney, catalyzes the conversion of enol- to keto-oxaloacetate by a mechanism in which solvent protons end up equally distributed between the two prochiral positions at C3 of keto-oxaloacetate. This conclusion is based upon the observation that when enzyme catalyzed ketonization is conducted in 3H2O in the presence of excess malate dehydrogenase and NADH, only 50% of the 3H in the isolated (2S)-[3-3H]malate is labilized to solvent upon treatment with fumarase. From a stereochemical perspective, this enzyme is unlike phenylpyruvate keto-enol tautomerase that is known to catalyze stereospecific proton transfer between solvent and the pro-R position of keto-substrate. As a result of an attempt to clarify the physiological importance of oxaloacetate tautomerase activity, keto-oxaloacetate was demonstrated to be directly transported across the inner membrane of rat liver mitochondria on the basis of the results of kinetic and isotope-trapping experiments.

zyme is unlike phenylpyruvate keto-enol tautomerase that is known to catalyze stereospecific proton transfer between solvent and the pro-R position of keto-substrate. As a result of an attempt to clarify the physiological importance of oxaloacetate tautomerase activity, keto-oxaloacetate was demonstrated to be directly transported across the inner membrane of rat liver mitochondria on the basis of the results of kinetic and isotope-trapping experiments.
Ho\ /c4HCo3+ (1) -0zc -ozc Phenylpyruvate keto-enol tautomerase and indolylpyruvate keto-enol tautomerase comprise the remaining known members of this general class of enzymes (2, 3). Although the physiological utility of oxaloacetate tautomerase has not been firmly established, the suggestion that this enzyme plays a crucial role in the metabolism of oxaloacetate is supported by the fact that tautomerase activity is widely distributed among plant, animal, and microbial extracts (1). Indeed, tautomerase activity is found in all compartments of the cell in which the metabolism of oxaloacetate occurs (4).
In order to evaluate the general stereochemical constraints on enzyme catalyzed tautomerization of a-ketoacids, the stereochemistry of the proton transfer reaction catalyzed by oxaloacetate tautomerase was determined for comparison with that of phenylpyruvate tautomerase, known to involve stere.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisemnt" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed. § Present address: Dept. of Physiological Chemistry, Ohio State University, Columbus, OH 43210.
ospecific exchange of the pro-R proton of keto-substrate with solvent (5). Contrary to expectation, enzymic ketonization of enol-oxaloacetate involves stereorandom incorporation of solvent protons at C3 of keto-oxaloacetate. Thus, oxaloacetate tautomerase must operate on the basis of a highly unusual stereomechanistic principle, provided the physiological role of this enzyme has been correctly assigned.

EXPERIMENTAL PROCEDURES
Materials-The following enzymes and substrates were purchased from Sigma: malate dehydrogenase (porcine heart), fumarase, NADH (grade 111) and oxaloacetic acid (grade I). The 3Hz0 and (S)-[U-14C] malate were purchased from New England Nuclear and Amersham-Searle, respectively. The (2S,3R)-[U-14C, 3-3H]malate was prepared by incubation of (S)-[U-14C]malate in 3Hz0 in the presence of fumarase and then purified by anion exchange (Dowex-C1) and by ion exclusion chromatography (see below). All other reagents were of the highest purity commercially available.
Analytical Methods-High performance liquid chromatography was carried out using a Water's Model 60QOA solvent delivery system equipped with a U6K sample injection system. Column effluent was monitored at 207 nm with an LKB Uvicord-S UV Monitor. Samples for liquid scintillation counting were prepared in Scint-A mixture (Packard) and counted on a Prias Liquid Scintillation Spectrometer. NMR spectra of solutions of oxaloacetic acid in &-dioxane were taken on an IBM NR-80 instrument. Kinetic measurements were carried out using a Gilford 2400-2 Spectrophotometer. Malate was quantitated by the malate dehydrogenase/hydrazine method (6). Transport kinetics of oxaloacetate into rat liver mitochondria were monitored using a Britton Chance Dua1:Wavelength Spectrophotometer cmstructed by Dr. Thomas Marsho (University of Maryland Baltimore County) according to specifications described elsewhere (7).
Purification and Assay of Oxaloacetate Tautomerase-The tautomerase from porcine kidneys was purified initially by the procedure of Annette and Kosicki (1) through the 40-60% (NH&S04 precipitation step to a specific activity of -3 units/mg. After dissolution of the precipitate in a minimal volume of Tris buffer (5 mM, pH 7) containing EDTA (0.1 mM) and dithioerythritol (0.01 mM), the protein solution was extensively dialyzed against the same buffer and then fractionated on two successive DEAE-cellulose columns equilibrated at different pH, (see Fig. 1). Protein concentrations were determined on the basis of an E;%nm = 2.4 ml/mg.cm.
Tautomerase activity was determined in the direction of ketonization using a coupled enzyme assay in which keto-oxaloacetate is trapped with excess NADH and malate dehydrogenase (1). Solid oxaloacetic acid, evaporated onto a small glass spoon from an ether stock solution (-0.05 pmol of enol-tautomer), was rapidly introduced into a cuvette containing 1 ml of potassium phosphate buffer (10 mM, pH 7.4), malate dehydrogenase (-30 units), and NADH (0.2 mM). After determining the nonenzymic ketonization rate on the basis of the slow decrease in absorbancy at 340 nm for 30 s, tautomerase (-0.05 units) was then introduced into the cuvette. Enzyme activity was calculated from the increase in the initial rate of loss of absorbancy and corrected to V, , units on the basis of the reported K,,, of 66 p M for enol-oxaloacetate (1).
Stereochemical Analysis-The strategy for determining the stereochemistry of the tautomerase reaction is depicted in Table I. In order to initiate the stereochemistry experiment, an aliquot of oxaloacetic acid in anhydrous dioxane (enriched in enol-tautomer) was introduced into a buffered solution containing tautomerase, excess 4535 malate dehydrogenase and NADH, as well as 3Hz0 and a trace amount of (S)-[U-'4C]malate. After incubation for 30 s (25 "C), the enzymes were removed by ultrafiltration through a PM-10 membrane and the filtrate adsorbed to a Dowex-C1 column (7 X 0.3 cm). The column was first washed with water to remove excess 3Hz0 and then the malate was eluted with aqueous HzS04 (15 mM). A portion of the pooled malate was further resolved by high performance liquid chromatography using an ion exclusion column (ORH-801 Organic Acids Column, %-inch outer diameter X 30 cm) purchased from Interaction Chemicals. Using aqueous HzS04 (5 mM) as an eluting solvent, the elution volume of the malate was -5 ml. The 3H/14C ratio for the isolated malate was then determined. The remaining pooled malate from the Dowex-C1 column was brought to dryness with a stream of dry air at 37 "C, dissolved in sodium phosphate buffer (5 mM, pH 7) and incubated with fumarase (23 units) for 30 min. The mixture was then ultrafiltered through a PM-10 membrane and resolved on the ion exclusion column as described above. Fumarate and 3Hz0 comigrate on the ion exclusion column with an elution volume of -8.5 ml and, therefore, are well resolved from malate. The 3H/14C ratio for the malate peak was determined and compared with that of the malate before treatment with fumarase in order to infer the stereochemistry of the tautomerase reaction.
Preparation of Mitochondria-The mitochondria used in the transport experiments were prepared from the livers of starved Sprague-Dawley rats by a modification of the "high-yield" procedure of Pederson et al. (8), in which 0.25 M sucrose was used in place of 0.22 M mannitol, 0.07 M sucrose. Acceptor control ratios were in the range 4-6. The mitochondria were loaded with phosphate according to the procedure of Passarella et ul. (9). Mitochondrial protein was determined by the Biuret method (10).

RESULTS
Purification and Molecular Weight of Oxaloacetate Tautomerase-In the isolation procedure described originally by Annette and Kosicki (l), oxaloacetate tautomerase was initially purified by heat, acetone, and (NH4)2S04 fractionation followed by column chromatography using hydroxyapatite prepared by the method of Massey (11). The reported highest specific activity was -5 units/mg. However, in our hands the column procedure, using commerical hydroxyapatite (Bio-Rad), typically resulted in poor yields of enzyme having -10fold lower specific activity than that of the enzyme preparation applied to the column (-3 units/mg).
Subsequently, DEAE-cellulose column chromatography was discovered to be an efficient and reproducible method for further purifying the dialyzed enzyme from the (NH4)&04 precipitation step. The successive use of two such columns, equilibrated at different pH, resulted in a four-fold increase in specific activity (Fig. 1). The phenylpyruvate tautomerase activity eluted in the pregradient wash of the second DEAEcellulose column, well separated from the oxaloacetate tautomerase activity. The pooled enzyme from fractions 50-62 of the second column was used in the stereochemistry experiment (specific activity = 11.5 units/mg). Attempts at further increasing the specific activity of the enzyme by gel-filtration chromatography and by affinity chromatography, using Sepharose-4-ethyl-oxaloacetate, were largely unsuccessful due to a marked decrease in the stability of the enzyme as extraneous protein is removed. The inclusion of dithioerythritol (0.01 mM) in all buffers used in the purification procedure marginally increased the stability of the enzyme.
The molecular weight of oxaloacetate tautomerase was estimated to be -55,000 by gel-filtration chromatography using a Sephadex G-200 column Composition of Oxaloacetic Acid in Dioxane Solution and in the Solid State-Determination of the Stereochemistry of enzymic ketonization involved introducing enol-oxaloacetic acid into a coupled enzyme system composed of tautomerase and malate dehydrogenase ( Table I). Since enol-oxaloacetic acid is capable of cis-trans isomerism, a means of introducing a single isomer of this tautomer to the coupled enzyme system had to be found in order to avoid ambiguities in the interpretation of the results of the stereochemistry experiment. Solid oxaloacetic acid was initially excluded for this purpose, given the uncertainty over the isomeric composition of the solid (15 and references therein).
A dioxane solution of oxaloacetic acid was finally selected as the source of the enol-tautomer, since a single isomer of this tautomer appears to predominate in dioxane solution on the basis of NMR measurements. In d,-dioxane, oxaloacetic acid exhibits a single, major vinyl proton resonance at 65.93 (Fig. 2). This accords with a previously published spectrum of oxaloacetic acid in the same solvent (15). The presence of significant concentrations of both cis and trans isomers should have been revealed by two well-resolved singlets due to the vinyl protons of each isomer with a separation of roughly 0.3 ppm. This prediction is based upon the difference in the chemical shifts of the vinyl protons due to the cis (65.96) and  Tetramethylsilane was present as a reference standard. Vinyl protons, due to enol-oxaloacetic acid, appear at 65.93; methylene protons, due to keto-oxaloacetic acid, appear at 63.78. The broad resonance at 68.7 is due to the OH protons of the tautomers. The resonance at 63.5 is due to protiodioxane. The inset shows the relative magnitudes of the vinyl and methylene proton resonances -15 min after preparation of the sample and, therefore, before equilibrium has been established between the enol-and keto-tautomers. trans (66.35) isomers of the model compound, [O-alkyllenoloxaloacetic acid, calculated on the basis of Pascual's shielding constants (16). Careful inspection of Fig. 2 reveals a minor resonance at 66.21 that could be attributed to the alternate isomer of the enol-tautomer. However, unlike the resonances at 65.93 and 63.79, the magnitude of this resonance was undiminished by the addition of an aliquot of 'HzO to the NMR sample, indicating that this resonance is not due to an exchangeable proton. Finally, although shielding constant calculations can be subject to substantial error, the above analysis is nevertheless indicative of the predominance of a single isomer of enol-oxaloacetic acid in dioxane solution. No claim is made as to which isomer accounts for the resonance at 65.93.
The discovery that the ketonization of enol-oxaloacetic acid is a slow process in da-dioxane solution provided a method for establishing that solid oxaloacetic acid is composed of the same isomer of the enol-tautomer that predominates in dsdioxane solution (Fig. 2, inset). The time-dependent decrease in the integrated intensity of the vinyl proton resonance after dissolution of solid oxaloacetic acid in da-dioxane is a first order process, hobs = 0.038 min" (32 "(2). Thus, the fraction of enol-tautomer present at the time of dissolution of solid oxaloacetic acid in ds-dioxane (A,) could be calculated from In ( [ A , -A,]/[A, -A,]) = -k&s. t where A, = per cent enoltautomer at any time t, and A , = percent enol-tautomer at t = m, corresponding to 68% enol-tautomer and 32% ketotautomer. Thus, A, was calculated to be 100 f 5%. Consistent with this conclusion is the fact that the calculated activity of oxaloacetate tautomerase is independent of whether the enzymic assay is initiated with solid oxaloacetic acid or with a solution of oxaloacetic acid in dioxane.
Nonenzymic Ketonization of Enol-oxaloacetate-In order to calculate the fraction of enol-oxaloacetate actually converted to keto-oxaloacetate by the tautomerase in the stereochemistry experiment, the rate constant for nonenzymic ketonization of enol-oxaloacetate had to be determined under the buffer conditions described in Table I. This was done by diluting an aliquot of oxaloacetic acid in dioxane solution (0.02 ml, 3.34 rmol), prepared 1 h before use, into buffer (2 ml) containing excess malate dehydrogenase (12 units) and NADH (6 mM) in a 1-cm cuvette and then following the time-dependent loss in optical density at 390 nm due to oxidation of the NADH.
The initial rapid loss in optical density was due to rapid reduction of the keto-tautomer, the direct substrate for malate dehydrogenase. This represented 29.3 & 5.7% of the total oxaloacetate present, in fair agreement with the results of the NMR experiment. The slower first order loss in optical density that followed was attributed to rate-limiting ketonization of enol-oxaloacetate, K = 0.40 k 0.01 min-l.
Stereochemistry of the Oxaloacetate Tautomerase Reaction-The stereochemistry of solvent proton incorporation into enol-oxaloacetate, .as catalyzed by the tautomerase in 3Hz0, was determined using a coupled enzyme reaction in  (Table  I). The coupled enzyme reaction was initiated using a stock solution of oxaloacetic acid in dioxane solution that had been prepared shortly (10 min T I I -1.

NADH NAD+
Greater than 95% of the enol-tautomer is calculated to undergo enzymic ketonization, given the initial concentration of enol  Table I, the inclusion of a trace amount of (S)-[U-14C]malate in the reaction mixtures allowed an accurate final assessment of the loss of 3H from the isolated (2S)-[3-3H]malate upon incubation with fumarase, on the basis of the decrease in the 3H/14C ratio. The data of Table I demonstrate that both enzymic and nonenzymic ketonization of enol-oxaloacetate in 3H20 results in the same (R,S)-ket0-[3-~H]oxaloacetate.

Permeability of Liver Mitochondria to Keto-oxaloacetate-
The unusual outcome of the stereochemistry experiment prompted an attempt to clarify the physiological role of the tautomerase in the cell. Heretofore, the significance of this enzyme activity has been unclear, since all of the oxaloacetatedependent enzymes so far examined use keto-oxaloacetate, the thermodynamically predominant tautomer (80-90%) under physiological conditions (18, 19). Thus, the conditions under which ketonization of the minor enol-tautomer would become physiologically important is uncertain. However, another oxaloacetate-dependent process whose tautomeric specificity has not been examined is the direct transport of oxaloacetate across the inner mitochondrial membrane. This process may be metabolically important under certain physiological conditions (20). Thus, if the translocator protein(s) involved in transport were specific for the enol-tautomer, the need for a tautomerase might be comprehensible, given that the keto-tautomer is the substrate for or the product of both intra-and extramitochondrial oxaloacetate-dependent enzymes.
That oxaloacetate is directly transported across the inner membrane of rat liver mitochondria in vitro was first suggested by the observation that exposure of mitochondria to exogenous oxaloacetate results in a rapid loss of absorbancy due to oxidation of intramitochondrial NAD(P)H, a process catalyzed by intramitochondrial malate dehydrogenase (21). Transport appears to limit the rate of oxidation, since this rate is greater in disrupted mitochondria. Subsequent in vitro studies demonstrated that transport is a saturable process obeying Michaelis-Menten kinetics (9,21,22). Transport may involve the so called "dicarboxylate" and/or "a-ketoglutarate" translocator systems (22,23).
In order to test whether enol-oxaloacetate is the exclusive substrate for transport, mitochondria were exposed alternately to keto-and enol-enriched samples of oxaloacetate and the comparative rates of, oxidation of intramitochondrial NAD(P)H followed by dual-wavelength spectrophotometry (Fig. 3). The observation that the initial rate of transport is greater using keto-enriched samples of oxaloacetate shows that the enol-tautomer is not the exclusive substrate for transport, according to the following scheme. containing defatted bovine serum albumin (1.9 mg/ml), 14 "C. After 10 s, the reaction mixture was quenched with 0.3 N HC10, (0.2 ml) and the denatured protein was spun down at 39,000 X g (15 min). After neutralizing the supernatant with 3 N KOH, the KC10, was removed by centrifugation and the malate isolated on a Dowexformate column (0.5 X 8 cm) using a linear formic acid gradient (0-5 N). The specific radioactivity of the transported oxaloacetate was calculated from Sp(Y) = Sp(Y),k ((Y+Z)/Y) where Sp(Y),k is the specific radioactivity of the malate eluted from the Dowex-formate column, 1.07 f 0.08 X lo5 cpmlpmol; Z = total endogenous malate present prior to transport, 3.00 -t 0.17 nmol/mg of protein; and Y = the additional endogenous malate produced in mitochondria exposed to oxaloacetate, 4.95 f 0.48 nmol/mg of protein.
*In order to determine the specific radioactivity of ket0-[3-~H] oxaloacetate (Sp(X)) prior to transport, the same experiment was done except that the reaction mixture was quenched by the addition of malate dehydrogenase (48 units) and NADH (7.5 pmol) followed by the addition of 3 N HClO, (0.2 ml). The magnitude of Sp(X) is equal to (Sp(X)
As a further test of the tautomeric specificity of transport, mitochondria were exposed to saturating levels (5 mM) of aqueous (R,S)-ket0-[3-~H]oxaloacetate. Transport was terminated after 10 s and the intramitochondrial (2S)- [3-3H] malate isolated (Chart I). That the transport of the ketotautomer is a direct process, not involving obligatory enolization, is indicated by the observation that the specific radioactivity of the intramitochondrial (2S)-[3-3H]malate (due to transport) is equivalent to that of the (R,S)-ket0-[3-~H]oxaloacetate used to initiate transport.

DISCUSSION
The central observation of this work is that oxaloacetate tautomerase catalyzes the ketonization of a single isomer of enol-oxaloacetate in 3H20 to give (R,S)-ket0-[3-~H]oxaloacetate.
Stereospecificity of Proton Transfer-Stereospecific proton transfer is the general rule among enzyme-catalyzed reactions, provided one excludes those enzymes for which an enol intermediate dissociates from the active site during catalysis (24). Nevertheless, two general explanations can be envisioned for the apparent lack of stereospecificity in the proton transfer reaction catalyzed by oxaloacetate tautomerase.
The first of these is based upon the failure to purify the enzyme to homogeneity. Conceivably, the preparation of oxaloacetate tautomerase used in the stereochemistry experiment contained two tautomerase species that catalyze ketonization with similar efficiencies but opposite stereochemistries. This would be an unprecedented explanation in that, to our knowledge, there are no other examples of two enzymes from the same biological source that catalyze the same reaction with opposite stereochemistries. Equally unprecedented is the possibility that a single tautomerase species contains two active sites that catalyze the same reaction with equal efficiencies but with opposite stereochemistries.
A second and seemingly more likely possibility is that enoloxaloacetate binds to the active site in such a way that both diastereotopic faces of the enol-tautomer are accessible to proton addition by the enzyme (two-base mechanism) or by solvent. A variation of this hypothesis would be obtained if there are different binding modes between substrate and enzyme so that the two diastereotopic faces of bound enoloxaloacetate alternatively undergo unidirectional protonation by the enzyme (single-base mechanism) or by solvent.
Relationship to Phenylpyruvate Keto-enol Tautomerase-Related to the question of mechanism is the more fundamental problem of why oxaloacetate tautomerase catalyzes a nonstereospecific proton transfer when the vast majority of enzymes that catalyze stereochemically cryptic reactions do so stereospecifically (24). The generality of stereospecific catalysis suggests that enzymes evolve partly in response to the need to achieve a high state of catalytic efficiency requiring a well-defined topological relationship between bound substrate and the catalytic residues within the active site. Phenylpyruvate tautomerase, an enzyme of the same reaction type as oxaloacetate tautomerase, conforms to this generality in that proton addition is to the 2si-3re face of the bound enoltautomer of substrate (5). Conceivably, stereospecificity is imparted by the presence of a catalytically important active site base positioned above this face of the bound substrate.
When viewed within this context, the absence of stereospecificity in the oxaloacetate tautomerase reaction is indeed surprising, although not unprecedented. The intermediate eneamine generated during the acetoacetate decarboxylase reaction has recently been reported to undergo nonstereospecific protonation on the way to the intermediate ketamine (25). Perhaps the catalytic efficiencies of the tautomerase and the decarboxylase do not depend on catalyzed protonation of substrate carbon, thus obviating stereospecific proton transfer.
Physiological Function of Oxaloacetate Tautomerase-At least in the case of the tautomerase, speculation about the catalytic mechanism must be tempered by the possibility that tautomerase activity is not a product of selective evolution, but rather is due to a fortuitous arrangement of amino acid residues that promote nonstereospecific proton transfer in a fashion analogous to that of a nonenzymic catalyst. Certainly, the requirements for catalysis are minimal in that acids, bases, and metal ions all serve as efficient nonenzymic catalysts of tautomerization (26)(27)(28). The decarboxylation of oxaloacetate is another example of a chemical process with minimal catalytic requirements (29)(30)(31)(32). Indeed, the oxaloacetate decarboxylase activity of pyruvate kinase may be an example of an aberrant property of an active site that has evolved for some other purpose (33,34). The same possibility must be carefully considered in the case of the tautomerase, although this enzyme is known to be distinct from the common oxaloacetate-dependent enzymes citrate synthetase, glutamate-oxaloacetate transaminase, malate dehydrogenase, and fumarase (1). In addition, commercial rabbit muscle pyruvate kinase (Sigma) does not exhibit detectable oxaloacetate tautomerase activity.
In a related vein, the physiological usefulness of tautomerase activity is unclear. Aqueous solutions of oxaloacetate near physiological pH are composed of roughly 80-90% keto-tautomer and approximately equal concentrations of enol-tautomer and hydrated oxaloacetate (18,19). However, all of the oxaloacetate-dependent biological processes so far examined use the predominant keto-tautomer as substrate or produce it as product. These processes include seven different enzymic reactions (1,35). In addition, the direct transport of oxaloacetate across the inner membrane of rat liver mitochondria involves the keto-tautomer (Fig. 3, Chart I). Since enoloxaloacetate has yet to be clearly identified as a biological substrate, the conditions under which tautomerization would influence the metabolism of oxaloacetate are unclear. In contrast, the biological function of phenylpyruvate tautomerase is less problematical, since the enzyme can use as substrate 4-hydroxy-3,5-diiodophenylpyruvate, the enol-tautomer of which may serve as the direct substrate for the thyroidal peroxidase in the biosynthetic pathway leading to thyroxine (36).
Solvent Tritium Isotope Effects-Even if tautomerase activity is an aberrant catalytic process, some degree of stereoselectivity during proton transfer would be expected by virtue of the fact that catalysis takes place on the asymmetric surface of a protein. In apparent contradiction to this expectation is the observation that enzyme-catalyzed ketonization of enoloxaloacetate gives rise to racemic ket0-[3-~H]oxaloacetate.
However, this observation does not prove that both diasteriotopic faces of the bound enol-tautomer are equally accessible to protonation, since the stereochemistry may be under ther-modynamic rather than under kinetic control.
This possibility is suggested from the comparative magnitudes of the primary solvent tritium isotope effects for enzymic versus nonenzymic ketonization of enol-oxaloacetate, calculated from the specific radioactivities of the isolated (2S)- [3-3H]malate with those of the 3Hz0 in which the reactions were conducted (Table I). For the nonenzymic case, proton transfer is at least partially rate-determining, k&T = ((10.88 f 0.01) X 104)/((1.48 f 0.11) X lo4 X 2) = 3.68 f 0.27.
The factor of two in the numerator of this equation accounts for the fact that ketonization involves the incorporation of a single solvent proton, whereas the specific radioactivity of the 3Hz0 is calculated on the basis of two exchangeable protons. For the~enzymic case, the isotope effect is near unity when calculated on the basis of the assumption that only one proton is incorporated from solvent: k &~ = ((12.46 f 0.08) X lo4)/ ((6.18 i~ 0.36) X lo4 X 2) = 1.01 f 0.06. This suggests that some other step besides proton transfer is rate-determining, possibly dissociation of keto-oxaloacetate from the protein.
Thus, the formation of racemic ket0- [3-~H]oxaloacetate by the tautomerase would be consistent with rapid (although possibly unequal) rates of equilibrium protonation of bound enol-tautomer to give either bound pro-R-or bound pro-Slabeled keto-[3-3H]oxaloacetate followed by rate-determining dissociation of product.
Conclusions-The proton transfer reaction catalyzed by oxaloacetate tautomerase is not stereospecific, in contrast to that catalyzed by phenylpyruvate tautomerase. The biological usefulness of oxaloacetate tautomerase activity is unclear. If indeed the tautomerase protein has evolved to specifically bind oxaloacetate, the biological significance of this interaction may be something other than to catalyze the tautomerization of oxaloacetate.