INTRODUCTION

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-Ketolases hydrolyze carbon-carbon bonds of 1,3-diketo substrates, and 10 of these enzymes are cited in the Enzyme Nomenclature listings (37) (EC 3.7.1.1 to EC 3.7.1.10). They are frequently encountered in microbial, plant, and animal cells. A brief survey of their occurrence has been provided recently (28). The role of -ketolases, like that of other hydrolases, e.g., proteases, lipases, and esterases, is clearly to form intermediary products suitable for processing by central metabolic pathways to carbon dioxide and to contribute to the biosynthesis of cellular constituents for growth or for detoxification. A few natural substances produce 1,3-diketo metabolites that are hydrolyzed by -ketolases to achieve C-C bond hydrolysis; these include the three aromatic amino acids, phenylalanine, tyrosine, and tryptophan. A carbon-carbon bond cleavage by kynureninase (EC 3.7.1.3) in tryptophan catabolism is a variant in that the formal "2-amino-4-keto" intermediate, kynurenine, is the substrate for the formation of anthranilate and alanine, which presumably occurs via its "2-imino-4-keto" derivative after condensation with the pyridoxal phosphate prosthetic group of the enzyme (1). The thymol, orcinol, and resorcinol catabolic pathways in different strains of Pseudomonas putida yield 2,4,6-triketo carboxylate intermediates after dioxygenative cleavage of hydroxyquinol substrates (7-9). The different compounds are hydrolyzed to carboxylates and 2,4-diketo carboxylates; orcinol and resorcinol are catabolized via acetopyruvate (Fig. 1A) (8, 9), and thymol is catabolized through 3-methylacetopyruvate (7), before further enzymic hydrolyses. Similar enzymic hydrolyses of vinylogous 1,5-diketones are common when these are formed after meta-ring opening of catechols and quinols in other catabolic pathways (2, 3, 11, 12, 24).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   (A) The acetopyruvate hydrolase reaction. (B) Main aqueous-solution structures of acetopyruvate (R = CH3) in phosphate buffer at pH 7.5. The proportions for each structural form are approximately as follows: [1]:[2]:[3] = 40:50:10. Interconversion rates are described by Guthrie (17) and given in the text. The structure shown for [2] (enol) is for the protonated enol form at pH 7.5.

Two -ketolase enzymes have been purified to apparent homogeneity: acetopyruvate hydrolase (EC 3.7.1.6) (10) from orcinol-grown P. putida and the fumarylacetoacetate hydrolase (EC 3.7.1.2) used in phenylalanine and tyrosine catabolism, from bovine liver (21). A molecular size of ~38 kDa was determined for the enzyme from P. putida (10). It was concluded that this enzyme was monomeric by sodium dodecyl sulfate (SDS) and native gel electrophoresis, molecular size exclusion chromatography, and ultracentrifugation (10). Data were not shown for the bovine liver enzyme (21). However, fumarylacetoacetate hydrolase accepted acetopyruvate, whereas acetopyruvate hydrolase from P. putida did not catalyze the hydrolysis of several -diketo acids such as fumarylacetoacetate. Acetoacetate, oxalate, and the product pyruvate were competitive inhibitors of the bacterial acetopyruvate hydrolase (10).

Acetopyruvate hydrolase from rat liver extracts (EC 3.7.1.5) was first described by Meister and Greenstein (Fig. 1A) (23). Although this enzyme was not extensively purified, Meister and Greenstein were able to show the hydrolysis of several diketo acids with longer-chain alkyl groups replacing the methyl group of acetopyruvate, indicating that 4-substituted 2,4-diketo acids possessed the structural prerequisites for binding at the active site(s); acetoacetone was not a substrate for hydrolysis for either of the liver enzymes or the inducible acetopyruvate hydrolase from P. putida. In our previous study of acetopyruvate hydrolase from P. putida (10), 2,4-diketocarboxylate substrate analogues were not available to us, and thus examination of the specificity was extremely limited. We have synthesized a variety of 2,4-diketo carboxylic acids with larger alkyl substituents attached to the 4-position, replacing the methyl substituent at C-4 with branched-chain, alicyclic, and arene substituents (5), and we now describe a spectrum of the substrate specificity of the bacterial enzyme.

The easiest and most rapid assay for acetopyruvate hydrolase is by UV. The 2,4-diketo acids have characteristic absorption maxima in the 285- to 340-nm range, depending on the individual compound (27). However, the hydrolysis products require individual analyses after derivatizations, extractions, separations, and measurements, e.g., by absorbance, high-performance liquid chromatography, or gas chromatography. Proton-nuclear magnetic resonance (NMR) measurements in situ, although relatively insensitive, offered an alternative assay for both reaction kinetics and stoichiometries with single reaction mixtures (Fig. 2). The value of ¹H-NMR enzymic assays in ¹H₂O is shown here.

View larger version (50K): [in this window] [in a new window]   FIG. 2.   1H-NMR stack plot of the acetopyruvate hydrolase-catalyzed cleavage of acetopyruvate (enol form, 2.15 ppm; keto form, 2.28 ppm; hydrate, 2.19 ppm) to acetate (1.88 ppm) and pyruvate (2.34 ppm) in the presence of Mn2+ ions. Spectra were taken every 13 min.

Previously, Guthrie (17) had analyzed the proton spectra of acetopyruvate and its protonated forms in organic solvents and aqueous buffers, pH 1.5 to 6.5, with continuous-wave spectrometers, 60 and 100 MHz. We have used routine one-dimensional (1D) pulse sequences with Fourier transform (FT)-NMR spectrometers and confirmed his observations and conclusions and at the higher pH values used for the enzyme assays. We used normal water as a solvent, and we used a presaturation pulse sequence to suppress the massive proton signals, which would otherwise saturate the analogue digital converter of FT spectrometers and thus conceal or distort some signals of interest in the 3.5- to 5.5-ppm region.

The substrates used were synthesized by Claisen condensations, and their structures were confirmed by the usual spectroscopic methods (5). Guthrie's experiments (17) showed that there are three main aqueous solution structures of the acetopyruvate carboxylate anion which are in equilibrium: 2,4-diketo, 4-keto-2-enol, and a 4-keto-2-hydrate (Fig. 1B). (Enol-enolate equilibrium at a pH of ~7.5 is discussed by Guthrie [17] and Brecker et al. [5]. We use the term "enol" to describe this here.) There are large differences in the proportions of these equilibrium structures, which are influenced by pH values, metal ions, and the nature of the terminal substituents (5). The substrate specificity of the -ketolase induced in P. putida is described. Some isotopic chemical and enzyme-catalyzed "virtual reactions" (1, 34) were examined. The main conclusion is that the aqueous-solution structures of different analogues, and not the formal diketo structure used to describe the pure crystalline solid or liquid forms of some analogues, are of paramount importance for the occurrence of enzymic hydrolysis. This was shown by ¹H-NMR experiments (5, 28, 30a).

	MATERIALS AND METHODS

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Bacterial and culture conditions. P. putida ORC (9) was grown in minimal medium (25) supplemented with glucose (20 mM), orcinol (5 mM), and Lab-Lemco (Oxoid) (0.1%) in a 14-liter fermentor (New Brunswick Scientific Inc.) for 16 to 24 h at 30°C. Lab-Lemco broth cultures (2 × 7 ml) were added to the medium defined above (2 × 200 ml), and the cultures were incubated at 30°C for 16 to 30 h, with shaking. These cultures were used to inoculate the fermentor (10 liters of medium) and were aerated at ~8 liters min¹ and stirred at 300 rpm. After the orcinol was exhausted (16 to 24 h), 1 M glucose (100 ml) and 0.5 M orcinol (100 ml) were added, and they were added again after ~10 to 16 h. Two to six further similar additions were made after the orcinol was used up, and the cultures were harvested and resuspended in 50 mM KH₂PO₄-NaOH buffer at pH 7.5, then stored at 14°C. Extracts were prepared by cell disruption using a French press (25).

Acetopyruvate hydrolase assays and purification. The routine rapid UV assay was used (10) for monitoring the purification procedure, which included two chromatographic separations on a hydrophobic-interaction chromatograph (XK 16; Pharmacia) and an ion exclusion column (IEC) (Q6; Bio-Rad); this is a modification of the previous procedure (10) to take advantage of the chromatographic supports presently available and fast protein liquid chromatography (FPLC). Crude cell extracts (supernatants from centrifugation at 105,000 × g) prepared in 20 mM phosphate buffer, pH 6.8, were treated with (NH₄)₂SO₄ after a protamine sulfate precipitation. The proteins that precipitated between 30 and 70% saturation were redissolved, dialyzed, and separated by FPLC on phenyl Sepharose (XK16; Pharmacia) by elution with a 0.0 to 2.0 M KCl stepwise gradient in phosphate, pH 6.8. An active fraction, eluted with 0.2 M KCl, was equilibrated in 10 mM imidazole buffer, pH 6.8, applied to an IEC (Q6; Bio-Rad), and fractionated with a 0.0 to 1.0 M linear KCl gradient in the buffer. Fractions 14 to 16 were examined by SDS-polyacrylamide gel electrophoresis (PAGE) (see Fig. 3), which indicated that one major protein was present. It did not catalyze the hydrolysis of acetopyruvate unless EDTA or Mg²⁺ ions were added (see Results and Table 2). An electrophoretically pure protein (SDS-gel electrophoresis) with a molecular size of ~38 kDa was obtained (purification, 69-fold; yield, 71%; specific activity, 4.8 µmol min¹ mg¹). The enzyme was stored at 4°C for immediate use and at 14°C for 2 to 6 months.

Chemicals. Medium chemicals and chromatographic supports were obtained from commercial sources. 2,4-Diketo acid analogues were synthesized as potential substrates and were fully characterized. Their aqueous-solution structures in equilibrium were characterized under a variety of conditions, including the effects of pH, divalent metal ions, and the unwanted side reaction of an intermolecular aldol condensation to produce dimers in 10 to 30 h during storage of the solutions at 20°C (5). We used ¹H-NMR and UV spectroscopy for these purposes.

¹H-NMR assays of acetopyruvate hydrolase reactions. All spectra were measured on a Varian Gemini-200 or on a Varian Unity-600 by using a 5-mm broadband probe head. The enzymic transformations and parallel measurements were carried out in a commercial NMR sample tube (outer diameter, 5 mm; length, 178 mm) rotating at 20 rps in the NMR machine. The substrates (20 mM) were dissolved in 100 mM aqueous phosphate buffer at pH 7.5 and added to the NMR sample tube (0.7 ml). A 20% (vol/vol) D₂O solution or a D₂O vortex capillary tube (22) was added for a lock signal. After a test measurement and field homogenization (shimming), the enzyme was added. The amount of enzyme was calculated so that complete cleavage of the substrate took place in 4 to 8 h. Then 18 spectra were recorded. For each spectrum, 128 scans were accumulated in about 13 min (200 MHz) or about 6 min (600 MHz). The spectra with the 4-tert-butyl substrate were taken with 16 scans (80 s) and no delay. All the spectra were referenced to the H₂O signal (4.70 ppm), and for the suppression of the water signal, the presaturation method (16, 20) was used for the 200-MHz NMR and the WET (water suppression enhanced through I₁ effects) method (26, 33) was used for the 600-MHz NMR. The following parameters were adjusted for a ¹H frequency of 200 or 600 MHz (5): a presaturation duration of 1.0 s (at 200 MHz), a ¹H pulse angle of 90°, an acquisition time of 2.0 s, and a relaxation delay of 1.5 s.

	RESULTS

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Acetopyruvate hydrolase. Acetopyruvate hydrolase was purified to apparent homogeneity from P. putida ORC by an alternative purification procedure (Table 1). It showed the same characteristics as the previous preparation from P. putida 01 (10). It was electrophoretically pure (by SDS-PAGE and PAGE) and was characterized as a monomeric protein with a molecular size of ~38 kDa (Fig. 3) and with no evidence of subunit structure in the native enzyme. Enzymic activity was enhanced by several divalent metal ions, e.g., Mg²⁺ and Mn²⁺, but was inhibited by Cu²⁺ ions (described below).

View larger version (65K): [in this window] [in a new window]   FIG. 3.   SDS-PAGE of purified acetopyruvate hydrolase and molecular size standards. Lane 1, crude cell extract; lane 2, after HIC; lane 3, SDS-PAGE standard proteins; lanes 4 to 6, sequential fractions 14, 15, and 16 after IEC. (Fractions were eluted with ~0.2 M KCl.)

The ¹H-NMR assay of enzymic activity. Guthrie (17) had clearly shown by ¹H-NMR that three major aqueous-solution forms of acetopyruvate exist at pH values of 1.5 to 6.5. We confirmed this with ¹H-NMR-FT spectra and obtained a pK_a of about 7.5 for the -keto-enol tautomers of the carboxylate anions (Fig. 4) (5, 17). This was important to show which solution structure was the actual substrate for the hydrolysis reaction (Fig. 1 and 2).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   1H-NMR spectrum of acetopyruvate (A) and two analogues (B and C) at pH 7.5.

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Comparison of the progress of acetopyruvate hydrolysis by the hydrolase with the 2H2O lock signal provided internally (A) or externally in a capillary tube (B).

Substrate specificity. Twelve substrate analogues were synthesized, and their aqueous-solution structures were unambiguously characterized (5). These analogue substrates gave a surprising spectrum of hydrolysis rates (Fig. 6). Acetopyruvate itself could be regarded as a very poor substrate; its rate of hydrolysis was ~20 times less than that for the 4-iso-propyl homologue (100%), while that for the 4-tert-butyl analogue was 68%. Even with a cyclohexyl substituent at the C-4, the hydrolysis rate was higher (25%) than that for acetopyruvate (4%). In contrast, analogues with 4-phenyl or 4-benzyl substituents were very poor substrates (<1%) (Fig. 6).

View larger version (37K): [in this window] [in a new window]   FIG. 6.   Substrate specificity of acetopyruvate hydrolase without added metal ions. Data are taken from the UV assays and were confirmed by the 1H-NMR method. Substrate concentrations for the two assays were 0.1 mM (UV) and 20 mM (NMR).

Effects of metal ions on hydrolase activity. Several bivalent metals markedly stimulated activity (Table 2). A Michaelis-Menten plot of the Mg²⁺ ion concentrations with acetopyruvate as the substrate gave apparent K_m and V_max values of ~1.7 mM and 4.8 µmol min¹ mg of enzyme¹ in the presence of Mg²⁺ ions. Mn²⁺ ions were more effective at lower concentrations than Mg²⁺ ions (Table 2) (10). After enzyme purification with the Cu²⁺ column (IEC), enzymic activity was not detected in collected fractions in the usual UV assay, but the addition of EDTA and several divalent cations to the assay mixtures restored activity. They presumably build a complex with Cu²⁺ ions eluted from the copper column. These observations were substantiated as shown in Table 2.

What is the aqueous-solution structure for hydrolysis? We needed a fast and more-sensitive ¹H-NMR assay to establish that it is the enol form which is hydrolyzed. 4-tert-Butyl-2,4-diketobutanoate (5,5-dimethyl-2,4-dioxohexanoate) (Fig. 4B) was used as the substrate for these experiments at concentrations of about 20 mM. The tert-butyl group has 9 equivalent protons and gives one singlet for each solution structure (enol, 1.08 ppm; diketo, 1.05 ppm; hydrate, 1.01 ppm). That is three times more sensitive than the methyl group signals in acetopyruvate. The three characteristic proton singlets were obtained in ~80 s, i.e., a spectrum could be displayed from 16 scans. Figure 7 clearly shows that the enol form was rapidly hydrolyzed first, during the first 300 s of the reaction time, while the concentrations of the diketo and hydrate structures remained constant. Thereafter, the enol and diketo forms disappeared at similar rates, while the hydrate was very slowly depleted (~10%). The experiment was repeated four times, showing this "burst" phenomenon, as with serine hydrolases (1, 13), followed by a rate determined by the rate constants for equilibrium of the two tautomers. As Guthrie showed, and we confirmed, the time to equilibrium of the keto-enol mixture is short ( = 0.1 s¹) compared to the time to equilibrium of the hydrate to the tautomers ( = 0.012 s¹) at pH 5 (Fig. 1) (5, 17). The relative rates of disappearance of the three forms are shown in Table 3, with data taken from Fig. 7.

View larger version (29K): [in this window] [in a new window]   FIG. 7.   Disappearance of the three structural forms of 4-tert-butyl-2,4-diketobutanoate and formation of pyruvate in aqueous solution.

	DISCUSSION

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Three main topics are discussed below: (i) the central role and ubiquity of -ketolases in general metabolic processes, mostly peripheral, (ii) the development of the proton-NMR method to monitor enzymic reactions in normal ¹H₂O and in ²H₂O, and (iii) the importance of understanding the aqueous-solution structures of substrates provided to enzymes and the preference shown for analogues of 2,4-diketobutanoates with large substituents at the C-4 position.

-Ketolases. It is clear that -ketolases (EC 3.7.1.[1 to 10]), which catalyze carbon-carbon bond hydrolyses, are frequently used in catabolic pathways (28). Carbon-carbon bond synthesis by these enzymes is energetically unfavorable in H₂O, and alternative thioester substrates with suitable leaving groups, e.g., CO₂, have been evolved to achieve this purpose. -Ketolases are ideal for catabolic processes once 1,3-diketo functionality has been introduced by oxidation reactions, and thus they represent an alternative to aldolases, thiolases, decarboxylases, oxygenases, etc., for fragmentation of the carbon skeletons of organic molecules. Most -ketolases described so far are intracellular enzymes, but for the biodegradation of polyvinyl alcohols, extracellular versions (EC 3.7.1.7) are produced by some microorganisms (28, 31). It is not known if any of these enzymes also catalyze retroaldol reactions of partially oxidized 1-hydroxy-3-keto intermediate polymers, which are undoubtedly formed initially by the extracellular oxidase(s) (18, 28, 31, 35). Like the expression of the intracellular -ketolases, that of the extracellular enzymic activities is specifically regulated by the provision of suitable precursor substrates, usually presented as growth substrates, that are oxidized to 1,3-diketones.

The proton-NMR method. The proton nucleus is present in almost all biological molecules. Proton-NMR spectra can be very definitive for diagnostic purposes and are useful for crude quantitative analyses (±5%), but their use for studies of this kind, to describe metabolic experiments in normal ¹H₂O, is rare in the literature (19, 28, 38). Experiments with nuclei other than protons have frequently been used to understand metabolic processes, including the transport of intermediates and ions across membranes and cells, since the 1970s (14, 25, 28, 32). We have found that the proton nucleus is very suitable for the analysis of enzymic reactions and those catalyzed by growing and nongrowing bacterial cell suspensions in situ (6, 19, 25, 30, 38). This is a relatively insensitive method, but it is entirely satisfactory when substrates, intermediates, and products are in high concentrations (1 mM) and the signals are not multiple nor coupled to neighboring protons or other nuclei in the substrates, although this also provides valuable information.

1

1

Aqueous-solution structures of substrates and acetopyruvate hydrolase specificity. The NMR experiments had shown that most 2,4-diketo acids exist in three main aqueous-solution structural forms at pH 7.5 (Fig. 1B) (5, 17): the carboxylate anions of 2,4-diketo, 2-enol-4-keto, and 2-hydrate-4-keto forms (~40:50:10). Exceptions to this were two arene analogues, 4-phenyl- and 4-benzyl-2,4-diketobutanoate (Fig. 4). They gave broad proton signals (~30 Hz line width at half-high) inconsistent with the structures assigned to their aliphatic and alicyclic counterparts. By using detailed 2D-NMR analyses (heteronuclear multiple quantum coherence and heteronuclear multiple bond correlation) it was suggested that a -delocalized electron distribution occurred between the 2- and 4-keto functions (Fig. 4) to form a quasi-six-membered ring with cations (5). Further support for this proposal was provided by the chelation complexes with divalent metal ions. Thus Mg²⁺ and Mn²⁺ ions formed chelates between the 2-enol and carboxylate anions of the aliphatic acids, whereas the two keto (2 and 4) oxygens (Fig. 4) formed a delocalized -electron system in conjugation with the arene substituents. In this respect the structures deduced for Cu²⁺ ion chelates entirely supported the two very different aqueous-solution structural forms of the alkyl and aryl analogues (5).

Structure of the substrates for hydrolysis. To determine the structures of the substrates used for hydrolysis, we had to conduct high-sensitivity proton-NMR experiments. Since the 4-tert-butyl analogue was a good substrate (Fig. 6) and could be used at relatively high concentrations, it was the choice for these experiments. The increased sensitivity for proton-NMR analysis afforded by this substrate gave data that led to the conclusion that the enolate-dianion structure was the preferred substrate for hydrolysis. The data shown in Fig. 7 suggest this, since the enol was consumed early in the reaction while the concentrations of the diketo and hydrate forms remained constant. Thereafter, the enol and diketo isomers disappeared at similar rates and the hydrate disappeared very slowly. This can be explained by the kinetic equilibrium constants for these molecular species (5, 17), in that the hydrolysis rate may become limited by the relatively fast keto-enol interconversions to equilibrium (17). These experiments required short accumulation times of 80 s for each spectrum to be obtained (16 scans) at the earliest times of the enzymic reactions (Fig. 7).

Divalent metal ions and hydrolase activity. We had previously shown that Mg²⁺ and Mn²⁺ ions markedly stimulated the enzymic hydrolysis of acetopyruvate and that Mn²⁺ was more effective than Mg²⁺ at 1/10 of its concentration (10). A chance observation was made during the purification of acetopyruvate hydrolase on a Cu complex column (IEC). Activity was not recovered in any eluted fraction, but when EDTA was added to reaction mixtures, the hydrolase was functional again. Some Cu²⁺-protein interaction seemed to have occurred, which was reversible by the counterion, EDTA, and was stimulated further by Mg²⁺ or Mn²⁺ ions. This was not investigated in detail.

View larger version (12K): [in this window] [in a new window]   FIG. 8.   Proposed coordination structures of Mg2+ and acetopyruvate (5) (A) and potential substrate binding to the enzyme (B).

General conclusion. A study of one enzyme of the -ketolase class of enzymes (EC 3.7.1.1 to EC 3.7.1.10) is insufficient for making broad generalizations, although some significant features do emerge from this work. Attention to, and understanding of the solution structures of these carbonyl substrates is very important for the interpretation of binding and catalytic events. We chose acetopyruvate hydrolase for its apparent simplicity but found quite unexpected complexity of the solution structures of several substrates and their metal chelates. Furthermore, we showed how acetopyruvate readily dimerizes in aqueous solution (5). The effect of the dimer formed (5) on the enzymic hydrolysis of monomeric substrates was not assessed, but it was shown that the dimer was not a substrate.