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J. Biol. Chem., Vol. 277, Issue 7, 4663-4671, February 15, 2002

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Effect of Metal Binding on the Structural Stability of Pigeon Liver Malic Enzyme^*

Hui-Chuan Chang, Wei-Yuan Chou, and Gu-Gang Chang

From the Graduate Institutes of Life Sciences and Biochemistry, National Defense Medical Center, Taipei, 114 Taiwan, Republic of China

Received for publication, November 21, 2001, and in revised form, December 4, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cytosolic malic enzyme from the pigeon liver is sensitive to chemical denaturant urea. When monitored by protein intrinsic fluorescence or circular dichroism spectral changes, an unfolding of the enzyme in urea at 25 °C and pH 7.4 revealed a biphasic phenomenon with an intermediate state detected at 4-5 M urea. The enzyme activity was activated by urea up to 1 M but was completely lost before the intermediate state was detected. This suggests that the active site region of the enzyme was more sensitive to chemical denaturant than other structural scaffolds. In the presence of 4 mM Mn²⁺, the urea denaturation pattern of malic enzyme changed to monophasic. Mn²⁺ helped the enzyme to resist phase I urea denaturation. The [urea]_0.5 for the enzyme inactivation shifted from 2.2 to 3.8 M. Molecular weight determined by the analytical ultracentrifuge indicated that the tetrameric enzyme was dissociated to dimers in the early stage of phase I denaturation. In the intermediate state at 4-5 M urea, the enzyme showed polymerization. However, the polymer forms were dissociated to unfolded monomers at a urea concentration greater than 6 M. Mn²⁺ retarded the polymerization of the malic enzyme. Three mutants of the enzyme with a defective metal ligand (E234Q, D235N, E234Q/D235N) were cloned and purified to homogeneity. These mutant malic enzymes showed a biphasic urea denaturation pattern in the absence or presence of Mn²⁺. These results indicate that the Mn²⁺ has dual roles in the malic enzyme. The metal ion not only plays a catalytic role in stabilization of the reaction intermediate, enol-pyruvate, but also stabilizes the overall tetrameric protein architecture.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pigeon liver malic enzyme ((S)-malate:NADP⁺ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) is a homotetrameric enzyme with a double dimer quaternary structure. It catalyzes the divalent metal ion-dependent reversible oxidative decarboxylation of L-malate to yield CO₂ and pyruvate with a concomitant reduction of NADP⁺ to NADPH. Cytosolic malic enzyme was first discovered in pigeon liver by Ochoa et al. (1) and was later found to be widespread in nature, from bacteria to human, in both cytosol and mitochondria. In animals and human, the major physiological function of the enzyme is in providing NADPH for the de novo biosynthesis of long chain fatty acids (2, 3). In tumor cells, the mitochondrial malic enzyme is involved in the glutamine metabolism. This provides an energy source for the malignant cells (4-6).

Rutter and Lardy (7) first characterized the requirement of an externally added metal ion in the catalytic mechanism of the malic enzyme. Detailed electron spin resonance and nuclear magnetic resonance studies have delineated the fundamental role of Mn²⁺ in the malic enzyme-catalyzed reaction (8). With the crystal structure of both mitochondrial and cytosolic malic enzyme having been solved (9-11), the role of the metal ion in the reaction mechanism of malic enzyme has become clearer. The metal ligands of the enzyme include Asp-258, Glu-234, Asp-235, C-1 carboxylate and C-2 hydroxyl of the substrate L-malate, plus a water molecule at the active site. The metal ion in the enzymatic reaction serves as a bridge between L-malate and the enzyme and functions to properly position the substrate L-malate at the active center as well as helping to polarize of the C-2 hydroxyl group in the initial stage. Subsequent decarboxylation of oxaloacetate is also catalyzed by the metal ion, which acts as a Lewis acid. The metal ion here plays a vital role in chelating the negatively charged enolate pyruvate intermediate (10, 12, 13). The effect of metallation on the structural integrity of the malic enzyme, however, has never been explored.

In the present work, we examined the urea-induced conformational changes of pigeon liver malic enzyme in the presence or absence of manganese ion. The structural role of metal ion was elucidated by utilizing various mutants with a defective metal binding ability.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Site-directed Mutagenesis-- Site-directed mutagenesis of pigeon liver malic enzyme was carried out according to the procedures of Kunkel et al. (14). The synthetic oligonucleotides used as mutagenic primers were: E234Q, 5'-CATTCAGTTTCAGGATTTTGC-3'; D235N, 5'-CAGTTTGAGAACTTTGCTAATG-3'; E234Q/D235N, 5'-CATTCAGTTTCAGAACTTTGC-3'; in which the mutation positions are highlighted and underlined in the oligonucleotide sequence. The pET21-ME recombinant phagemids were amplified in the ung and dut CJ236 Escherichia coli strain with helper phage R408 for preparation of the uracil-containing DNA template. The latter was then annealed with phosphorylated mutagenic oligonucleotides and in vitro extended and ligated by T4 DNA polymerase and T4 DNA ligase, respectively. The mutated DNA was screened by transforming it into the ung⁺ and dut⁺ JM109 E. coli strain, and the surviving colonies were further identified by dideoxy chain termination sequencing (15). The entire cDNA was also sequenced to exclude any unexpected mutations resulting from the in vitro DNA polymerase extension.

Expression and Purification of the Recombinant Pigeon Liver Malic Enzymes-- The wild type and mutated plasmids were transformed into BL21 bacteria. The transformants were incubated in LB medium and induced by 1 mM isopropyl -D-thiogalactopyranoside (IPTG) and then cultured overnight. The cells were then harvested by centrifugation at 5,000 × g for 10 min. Following sonication, the recombinant enzymes were purified by two chromatographic steps according to Chou et al. (16). When subjected to SDS-PAGE, all purified enzyme preparations show a single protein band with a molecular weight of ~62,000. This is consistent with the molecular weight (62,061) calculated from the amino acid sequence of the enzyme subunit (17). The recombinant WT¹ malic enzyme has an identical amino acid sequence and thus has the same physicochemical properties as those of the natural malic enzyme as characterized previously (16).

Enzyme Assay and Protein Determination-- Malic enzyme activity was assayed according to the published procedure (16). Protein concentration was determined by the protein-dye binding method (18). Apparent Michaelis constants for the substrate and cofactors were determined by varying the concentration of one substrate (or cofactors) around its K_m value and maintaining the other components constant at the saturation level. The kinetic parameters were obtained by fitting experimental data to appropriate kinetic models. The calculation was carried out with the EZ-FIT (19) or Sigma Plot 5.0 program (Jandel, San Rafael, CA).

Enzyme Denaturation in Urea Solution-- The urea solutions were freshly prepared daily. WT or mutant malic enzymes preincubated with or without 4 mM Mn²⁺ were denatured with various concentrations of urea in Tris acetate buffer (0.1 M, pH 7.4) at 25 °C for 1 h. The unfolding of the enzyme was monitored by fluorescence, circular dichroism (CD) spectra, and enzyme activity loss. For the equilibrium urea denaturation experiments, the fluorescence spectrum was also recorded at various times between 0 and 3 h to detect any time-dependent spectrum change. One hour of incubation was found to be enough for the denaturation to reach equilibrium.

Fluorescence spectra of the recombinant malic enzymes were analyzed with a Perkin-Elmer LS 50B luminescence spectrometer at 25 °C. All spectra were corrected for the buffer absorption. The Raman spectrum of water was also corrected. Both the red shift and the fluorescence intensity changes were analyzed together using the average emission wavelength method (cf. 20). The average emission wavelength (<>) was calculated according to Equation 1,

(Eq. 1)

in which F_i is the fluorescence intensity at the specific emission wavelength (_i).

CD measurements were made with a Jasco J-810 spectropolarimeter using a 0.1-cm path length cell and averaging the three repetitive scans between 250 and 200 nm. Parallel spectra of urea without protein were also recorded and subtracted from the sample spectra. Mean residue ellipticity () was obtained by Equation 2,

(Eq. 2)

in which 111.42 was used for M_MRW, the mean amino acid residue weight. d is the cell path in cm, and c is the enzyme concentration in mg/ml.

In the enzyme activity measurements, control experiments were performed simultaneously with the same amount of urea added into the assay mixture.

Heat Stability-- The sensitivity of WT and various mutant malic enzymes to thermal denaturation was examined by CD, for which an automatic programmable thermal control of the circulating water bath was used (Neslab, model RTE 111, Newington, NH). The enzyme in borate buffer (100 mM, pH 7.4) was preincubated with or without 4 mM Mn²⁺, and the enzyme solution was heated from 30 to 95 °C with a 0.5 °C interval increment. The enzyme conformational change was monitored by ellipticity at 222 nm.

Quenching of Pigeon Liver Malic Enzyme by Acrylamide-- Quenching titrations with acrylamide were performed at 25 °C by sequentially adding aliquots of the concentrated quencher stock solution (6 M, pH 7.4) to the enzyme solution. The excitation wavelength was set at 295 nm and the fluorescence emission spectra were scanned from 300 to 400 nm. The integration area between 330 and 360 nm was used for data analysis. The inner filter effect due to acrylamide absorption was corrected according to the method of Calhoun et al. (21).

The fluorescence quenching data in the presence of acrylamide was analyzed by the Stern-Volmer equation shown in Equation 3 (22),

(Eq. 3)

in which F_o and F are the fluorescence intensities in the absence or presence of the quencher, respectively. K_sv is the dynamic Stern-Volmer quenching constant, and [Q] is the quencher concentration.

When the Stern-Volmer plot displayed an upward curvature, the static quenching concept was used and the experimental data were fitted to a revised Stern-Volmer equation shown in Equation 4 (22),

(Eq. 4)

in which V is the static quenching constant measuring the complex formation between acrylamide and the enzyme.

ANS Binding Measurement-- Binding of ANS with the unfolded enzyme was accessed by measuring the fluorescence enhancement of ANS. The excitation wavelength was set at 370 nm, in which ANS has an extinction coefficient of 5,620 M¹ cm¹ (23). The emission spectra were integrated from 420 to 560 nm. All measurements were corrected for the background intensity of the buffer.

Analytical Ultracentrifugation Analysis-- The molecular mass of the enzyme under various conditions were estimated by a Beckman-Coulter XL-A analytical ultracentrifuge with an An60Ti rotor. Sedimentation velocity was performed at 20 °C and 40,000 rpm with standard double sectors aluminum centerpieces. The UV absorption of the cells was scanned every 5 min for 2 h. The data were analyzed with the SedFit program (24). Sedimentation equilibrium was performed at 20 °C with six-channel epon centerpieces and then centrifuged at 12,000 rpm for 12 h. The data was analyzed with software provided by Beckman-Coulter. The solvent density and viscosity in the presence of urea were corrected with the UltraScan II (website: www.ultrascan.uthscsa.edu/). The partial specific volume of malic enzyme was 0.7403 (25). All samples were visually checked for clarity after ultracentrifugation, and no indication of precipitation was observed.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fluorescence and Circular Dichroism Spectra of Pigeon Liver Malic Enzyme-- The intrinsic fluorescence is a sensitive probe to monitor the conformational change of a protein. We have examined the fluorescence of the native malic enzyme (26). Almost identical results were obtained with the recombinant malic enzymes. When excited at 280 nm, the recombinant WT malic enzyme showed a fluorescence spectrum with a maximum at 324 nm (Fig. 1A, closed circles). After denaturing with urea, there was a large red-shifting of the fluorescence to 356 nm (Fig. 1A, open triangles). The fluorescence intensity decreased as the urea concentration increased. Under the same conditions but excited at 295 nm, the maximum emission wavelength of the enzyme changed from 333 to 359 nm (not shown). Analysis of the secondary structure of the enzyme by CD spectrum also showed a decreasing signal as the urea concentration increased (Fig. 1B). The fluorescence and CD signals thus provide excellent tools in monitoring the conformational changes of malic enzyme during the unfolding process.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Emission fluorescence and CD spectra of pigeon liver malic enzyme. A, enzyme (27.6 µg/ml) was treated with or without urea, and the fluorescence emission spectrum was monitored with an excitation wavelength of 280 nm. B, CD spectra from 200 to 240 nm of the enzyme (465 µg/ml) treated with or without urea. The urea concentrations were: 0 (closed circles), 4.5 M (closed squares), and 9.4 M (open triangles).

Urea Induced Unfolding Process of Pigeon Liver Malic Enzyme-- When the enzyme was incubated with various concentrations of urea and the unfolding reached an equilibrium, a gradual red shift of the fluorescence spectrum, change of CD spectrum, and loss of enzymatic activity were observed (Fig. 2A). Both the shifting of fluorescence wavelength and ellipticity at 222 nm with varying urea concentrations indicated a highly reproducible biphasic denaturation curve, suggesting a three state-unfolding model. The [urea]_0.5 corresponding to the N  I and I  U processes were estimated to be 3.1, 5.6, and 3.4, 5.5 M, respectively, for the fluorescence and CD data. The integrated area of the fluorescence profile showed a steady state in low urea concentrations (<1.2 M), followed by a decreasing signal along with the inactivation of enzyme activity. Interestingly, there was a peak between 2.5 and 6 M urea, which coincided exactly with the transition region of the biphasic phenomenon observed in the fluorescence and CD spectra as described above.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Biphasic denaturation of pigeon liver malic enzyme induced by urea. The enzyme (27.6 µg/ml) without (A) or with (B) preincubation of 4 mM MnCl2 was mixed with various concentrations of urea at 25 °C for 1 h. The circular dichroism signal at 222 nm (open squares), the average fluorescence emission wavelength (open triangles), the enzyme activity (open circles), and the integrated fluorescence area (closed circles) were plotted versus denaturant concentration.

When the enzyme denaturation was monitored by enzyme activity loss, concentrations of urea of less than 1.2 M activated the enzyme activity. The [urea]_0.5 corresponding to the activation and inactivation processes were estimated to be 1.0 and 2.2 M, respectively.

Effect of Metal Binding on the Urea-induced Unfolding of Pigeon Liver Malic Enzyme-- The malic enzyme catalyzed reaction is metal-dependent with Mn²⁺ as the most effective cofactor. When malic enzyme was preincubated with an excess of Mn²⁺ and then treated with various concentrations of urea, there was a remarkable change of the unfolding pathway as shown in Fig. 2B. The unfolding of the malic enzyme shifted from a biphasic three-state phenomenon to a sigmoid two-state process detected by both fluorescence and CD measurements. The prominent peak observed around 4-5 M urea by the integrated fluorescence area also disappeared (Fig. 2B, closed circles). The [urea]_0.5 corresponding to the change of fluorescence wavelength and 222 nm ellipticity were estimated to be 4.5 and 4.7 M, respectively. The enzyme activity was protected by Mn²⁺. [Urea]_0.5 shifted from 2.2 (Fig. 2A) to 3.7 M in the presence of Mn²⁺ (Fig. 2B).

Metal Effect on the Mutant Malic Enzymes with a Defective Metal Binding Site-- The above results indicate that Mn²⁺ increases the structural stability and prevents the formation of an intermediate form during urea denaturation. To further characterize the role of metal ion in the structural stabilization of the malic enzyme, some of the metal binding ligands of pigeon liver malic enzyme were mutated to eliminate the metal effect.

The metal binding ability of E234Q was decreased by ~100-fold whereas the D235N or E234Q/D235N had little effect on K_m,Mn and K_m,Mal. The K_m,NADP values for all mutants were indistinguishable from that of the WT enzyme. On the other hand, the catalytic constants (k_cat) were reduced by 754-, 117-, and 2752-fold, respectively, for the E234Q, D235N, and E234Q/D235N mutants. These results indicated the importance of Glu-234 and Asp-235 in the catalytic mechanism of malic enzyme as suggested by the crystal structure (11).

In the absence of Mn²⁺, the mutants E234Q, D235N, and E234Q/D235N displayed a similar urea-induced biphasic unfolding pathway with that of the WT enzyme as monitored by CD spectroscopy (Fig. 3). However, unlike the WT, when the mutant malic enzymes were preincubated with 4 mM Mn²⁺, a biphasic unfolding phenomenon persisted and the denaturation curves almost overlapped with the curves without any Mn²⁺ being added. Comparing the average emission wavelength of the intrinsic fluorescence of the mutants yielded the same results (Fig. 4). Finally, we examined the unfolding pathway of the mutants by the integrated fluorescence area (Fig. 5). The characteristic peak observed between 2.5 and 6 M urea, disappearing in the presence of Mn²⁺ for the WT, did not change with the mutants E234Q and D235N. This characteristic peak, however, became less prominent in the single mutants and disappeared in the E234Q/D235N double mutant (Fig. 5D).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Comparison of the urea denaturation profiles of wild type and mutant pigeon liver malic enzymes by the mean residue epllipticity. The wild type (A) and mutants (B, E234Q; C, D235N; and D, E234Q/D235N) were preincubated with (closed circles) or without (open circles) 4 mM Mn2+ and then mixed with various concentrations of urea at 25 °C for 1 h. The mean residue ellipticity at 222 nm was measured to monitor the secondary structural change of the enzyme. The enzyme concentration was 0.465 mg/ml in all cases.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Average emission fluorescence wavelength changes of the wild type and mutant pigeon liver malic enzymes during urea denaturation. The wild type (A) and mutants (B, E234Q; C, D235N; and D, E234Q/D235N) were treated with Mn2+ and urea under the same conditions as described in Fig. 3. The enzyme concentration was 27.6 µg/ml. The average emission wavelength was collected at an excitation wavelength of 280 nm.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Comparison of the fluorescence integrated area changes of the wild type and mutant pigeon liver malic enzymes. The wild type (A) and mutants (B, E234Q; C, D235N; and D, E234Q/D235N) were treated with Mn2+ and urea under the same conditions as described in Fig. 3. The fluorescence emission integrated area between 320 and 370 nm was calculated.

The thermostability of these mutants was similar to that of the WT enzyme (Fig. 6). All recombinant malic enzymes had a T_m value of around 56 °C. Mn²⁺ had a significant impact on the secondary structure of the enzyme as revealed by the CD spectra.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Thermostability of WT and mutant malic enzymes. The wild type (A) and mutants (B, E234Q; C, D235N; and D, E234Q/D235N) preincubated with (closed circles) or without (open circles) 4 mM Mn2+ were heated in a programmable constant temperature water bath and monitored for CD spectral change.

Conformational Characterization of the Intermediate State of Malic Enzyme during Urea Denaturation-- The above results clearly indicated that an intermediate state was detected during the urea denaturation of the malic enzyme. To further characterize this intermediate state, the intrinsic fluorescence of the enzyme was examined by quenching it with acrylamide. This is a non-selective quencher of protein molecules that can penetrate into the interior of the protein matrix (21). WT malic enzyme gave linear Stern-Volmer plot with or without Mn²⁺ (Fig. 7A).

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Stern-Volmer plot for quenching of the intrinsic fluorescence of pigeon liver malic enzyme by acrylamide. Pigeon liver malic enzyme (177 µg/ml) was preincubated with (closed circles) or without (open circles) 4 mM Mn2+ and denatured with 0 M (A), 1 M (B), 2.5 M (C), 4.5 M (D), and 6 M (E) urea at 25 °C for 1 h. The denatured enzymes were then titrated with various amounts of the acrylamide. The excitation wavelength was set at 295 nm. The emission of the fluorescence-integrated area between 320 and 370 nm was calculated. The dilution factor and inner filter effect due to acrylamide absorption at 295 nm were corrected. The data were analyzed according to the Stern-Volmer equation (Eqs. 3 or 4).

At 1 M urea, in which the enzyme activity was activated, a slight upward curve Stern-Volmer plot indicated conformational differences that were prevented by Mn²⁺. At 2.5 M urea, where the fluorescence and CD spectra were insensitive to the conformational changes of the enzyme, a large quenching constant clearly indicated the structural difference. Mn²⁺ provided complete protection against this conformational change (Fig. 7C). The conformational changes induced by a further increase in urea concentration were only partially prevented by Mn²⁺ (Fig. 7, D and E). The various quenching constants are summarized in Table I.

The exposure of the hydrophobic area of the enzyme during urea denaturation was accessed by measuring the ANS binding. ANS binds with a high affinity to hydrophobic patches, which enhances the fluorescence of the reagent (27). Fig. 8 shows a clear peak corresponding to the intermediate state and indicates the hydrophobic nature of this state.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Binding of ANS to pigeon liver malic enzyme during urea denaturation. The wild type enzyme (126 µg/ml) with (closed circles) or without (open circles) Mn2+ preincubation was unfolded with various concentrations of urea at 25 °C for 1 h and the fluorescence emissions were measured after ANS addition. The excitation wavelength was set at 370 nm. The integrated fluorescence area between 420 and 560 nm versus urea concentration data was presented. The final concentration of ANS was 50 µM.

Quaternary Structural Changes of the Malic Enzyme During Urea Denaturation-- The quaternary structural changes of the malic enzyme were examined by analytical ultracentrifugation. The WT tetrameric malic enzyme has a molecular mass of 248 kDa, and sediments at 9 S as a single peak (Fig. 9A), in agreement with the value reported in the literature (25). In 6 M urea, the 9 S peak disappeared and was displaced by low molecular weight species. The intermediate state observed at 4-5 M urea did not show a discrete peak. Instead, there were species with S-values from 5 to 25 (Fig. 9B). These results clearly indicated polymerization of the enzyme. In the presence of 4 mM Mn²⁺, the polymer forms disappeared, and most of the enzyme existed as dimers (Fig. 10). Including 12 mM NaCl in the enzyme solution gave exactly the same result as the control (no metal added). The specific effect of Mn²⁺, therefore, is not that of salt. The randomly distributed residual values shown in the middle panels of Fig. 9 indicated an acceptable model for the sedimentation velocity experiments. We thus explored the sedimentation coefficient distribution in the whole range of urea denaturation for the malic enzyme (Fig. 10).

View larger version (40K): [in this window] [in a new window]   Fig. 9.   Sedimentation velocity experiments of pigeon liver malic enzyme. A, native enzyme. B, 4 M urea-denatured malic enzyme. The experimental details are described in the "Experimental Procedures." The enzyme concentration was 0.64 mg/ml. The three panels in each experiment represented the trace of absorbance at 280 nm during the sedimentation, the residues of the model fitting, and the sedimentation coefficients distribution of all species.

View larger version (29K): [in this window] [in a new window]   Fig. 10.   Quaternary structural changes of pigeon liver malic enzyme during urea denaturation. The enzyme (0.64 mg/ml) without (open circles, scales at the left axis) or with (connected solid lines, scales at the right axis) preincubation of 4 mM Mn2+ was mixed with various concentrations of urea at 25 °C for 1 h and then examined for quaternary structural changes by the sedimentation velocity. Only the distribution of the S values is reported. From A to H, the urea concentrations used were: 0, 1, 2, 3, 4, 4.5, 5, and 6 M, respectively. Please note that some of the panels have an enlarged y axis scale to highlight the distribution of various polymerization forms. For best results, the rotor speed and sedimentation duration were set at 40,000 rpm for 2 h (panels A and B) or for 5 h (panel C); 15,000 rpm for 5 h (panels D-G); and 50,000 rpm for 6.6 h (panel H).

WT malic enzyme sedimented at 9 S with or without Mn²⁺ in the solution (Fig. 10A). In the presence of urea, the tetrameric peak shifted to lower S values and a monomeric peak appeared in the absence of Mn²⁺. Manganese ion could protect the enzyme from dissociation (Fig. 10, B and C). At urea concentrations starting from 3 M, the enzyme polymerized. This was prevented by Mn²⁺ in the whole range of the phase I transition (Fig. 10, D-F). The second phase of the denaturation process involved a depolymerization and unfolding of the monomers, which was not protected by Mn²⁺ (Fig. 10, G and H).

Polymerization of the enzyme in the intermediate state was also demonstrated by sedimentation equilibrium analysis (Table II), which registered the average molecular weight of all species at a specific urea concentration. The E234Q, D235N, and E234Q/D235N mutants had molecular mass distribution similar to that for the WT (not shown). Due to the heterogeneity of the species, no further modeling was attempted for the sedimentation equilibrium data.

We have also tested the protection of the malic enzyme from aggregation in urea solution through different concentrations of Mn²⁺. Mn²⁺ at 2 or 40 µM provided no protection, but 400 µM Mn²⁺ provided substantial protection. Mg²⁺ at 4 mM provided partial effect, but 100 mM Mg²⁺ gave a similar protective effect as that of 4 mM Mn²⁺ (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have demonstrated a metal ion-induced slow conformational change of the malic enzyme, which toggled the enzyme between the R-state and the T-state with different enzyme activities (28). More recently, we found that Lu³⁺ also induced a slow isomerization of the human mitochondrial malic enzyme and transformed the enzyme into a dead-end inactive conformation.² Mn²⁺ was able to regenerate the native conformation and fully recover the inhibited enzyme activity. In other word, Mn²⁺ is required in maintenance a catalytically competent conformation of the enzyme. In corroboration with these results, the data shown in the present paper also indicate that the metal ion not only plays an essential role in the catalytic mechanism but also has a great impact on the structural features of the enzyme. In the absence of metal ion, the enzyme was polymerized at an intermediate concentration of urea. Metal ion provides substantial protection against polymerization. The most obvious protective effect of Mn²⁺ located at 3-4 M urea is in the intermediate state region (Fig. 10, D and E). Malic enzyme possesses a double dimer quaternary structure with two kinds of subunit interactions (9, 11, 29). The major form of malic enzyme observed in the presence of Mn²⁺ in the intermediate state is the dimeric form, which should be in a partially unfolded state. If the partially unfolded dimer intermediate has a tendency to aggregate, then it is conceivable that a tetrameric structure is the most stable form of the enzyme in the solution. The physiological or pathological meaning of this observation is not clear at this point in time. The polymer forms may not accumulate under the physiological conditions in which the manganese and magnesium concentration are in the µM and mM ranges, respectively.

It should be noted that there are significant protein dynamic quenching constant changes (Fig. 7) and detectable fluorescence area changes (Fig. 2) proceeding to the CD changes in the phase I transition (Fig. 2). Furthermore, ANS appears to bind to this state (Fig. 8). These properties are characteristic for a classic molten-globule state (30, 31). It is possible that, at the phase I transition, some portions of the enzyme structure are becoming molten-globule-like and that this conversion is modulated by metal binding. Similar cases have been demonstrated for a number of other large proteins (see Ref. 32).

The results shown in Figs. 2-5 seem to suggest that malic enzyme was unfolded at urea concentrations greater than 6 M. The highest urea concentration used in this study is 9.4 M in which the emission fluorescence spectrum is already 356 nm, close to that observed for an indole in an aqueous environment. However, even at 9.4 M urea, the enzyme molecules may not necessarily represent a completely unfolded form (see Ref. 23). Any structural changes beyond 9.4 M urea will not be revealed by the techniques used in this study.

It is quite common for the metal ion to play both catalytic and structural roles in an enzyme molecule. Among the goals of enzyme engineering is to try to improve the enzyme stability by introducing a de novo metal site into the enzyme or by modifying the existing one (33-36). Manganese is found in many redox and hydrolytic metalloenzymes. Enzymes that require manganese ion canonically adopt a basic , /, or //-fold (37). In all known cases, the manganese binding sites are located within or near the core -sheet structural elements. The resolved crystal structure of the malic enzyme reveals that each monomer of the enzyme is composed of four structural domains (9-11). Domain C contains the common Rossmann fold, which binds the nucleotide. Domain B belongs to a new fold for oxidoreductases (9). Both domains B and C adopt an overall // scaffolding. The metal binding site is located at the interface of the two central -sheets of domains B and C. Binding of the metal ion to the enzyme induced rearrangement at the interface regions and thus changed the subunit association affinity (38). The contribution of manganese ion in dual catalytic and stability roles has been proven in several other cases (39-42) and may be a general case.

The novel part of this paper is that we demonstrate, for the first time, the structural role of Mn²⁺ in pigeon liver malic enzyme. Mn²⁺ not only stabilizes the native tetrameric structure, but also prevents the polymerization of the partially unfolded enzyme, which is prone to aggregation due to the exposure of hydrophobic interiors as manifested by increased ANS binding (Fig. 8). The ANS binding studies, however, cannot exclude any ionic interactions between ANS and the enzyme molecule. The anionic nature of ANS molecule may also interact with the cationic sites of proteins (42). The active site integrity also needs Mn²⁺ (Fig. 2). At the same concentration, Mn²⁺ provides a better stabilization effect than Mg²⁺. There are at least two orders of magnitude of difference between the K_m values for Mn²⁺ and Mg²⁺. Malic enzyme might thus have evolved with a preference for the transition metal manganese over the alkaline earth metal magnesium due to its chemical nature. Manganese, being classified as a hard cation, has lower polarizability and a greater charge than magnesium. A hard cation will prefer hard ligands, e.g. the negatively charged oxygen atoms of aspartate, glutamate, or the solvent water (43). The metal ligands of malic enzyme as revealed by x-ray structure and biochemical analysis (11, 44-45) fulfill this prediction.

Direct involvement of Glu-234 and Asp-235 as the metal ligands has been demonstrated in the x-ray structure of both cytosolic and mitochondrial malic enzymes (10, 11). The effects of the mutations of Glu-234 and Asp-235 on the K_m,Mn and k_cat were not as large as expected for the essential groups. This could be due to the fact that Mn²⁺ has an octahedrally coordinated structure in the active site and has six ligands attached. Removing one or two of the ligands does not preclude the metal binding ability of the other ligands. Alternatively, the mutations could simply induce a conformational change that favors the participation of vicinal potential ligands, i.e. Asp-257 or Asp-141, in metal binding and compensates for the lost binding energy. Because the pigeon malic enzyme already has a crystal structure that supports the involvement of Glu-234 and Asp-235 as the metal ligands (11), our results provide clear indications of the sophistication of enzyme catalysis and the functional plasticity of the enzyme active site (46).

	ACKNOWLEDGEMENT

We thank Daniel L. Floyd for reading the manuscript before submission.

	FOOTNOTES

^* This work was supported by the National Science Council, ROC (Frontiers in Sciences Program, NSC 90-2321-B016-001). A preliminary report has been presented at the Fifteenth Symposium of the Protein Society held at Philadelphia, Pennsylvania from July 28 to August 1, 2001.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 886-2-87923100 (Ext. 18833); Fax: 886-2-29339996 or 886-2-87921544; E-mail: ggchang@ndmctsgh.edu.tw.

Published, JBC Papers in Press, December 5, 2001, DOI 10.1074/jbc.M111156200

² C. W. Kuo, H. C. Hung, L. Tong, and G. G. Chang, unpublished results.

	ABBREVIATIONS

The abbreviations used are: WT, wild type; ANS, 1-anilinonaphthalene-8-sulfonic acid; CD, circular dichroism.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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