Báo cáo khoa học: Functional role of fumarate site Glu59 involved in allosteric regulation and subunit–subunit interaction of human mitochondrial NAD(P)+-dependent malic enzyme pptx

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Báo cáo khoa học: Functional role of fumarate site Glu59 involved in allosteric regulation and subunit–subunit interaction of human mitochondrial NAD(P)+-dependent malic enzyme pptx

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Functional role of fumarate site Glu59 involved in allosteric regulation and subunit–subunit interaction of human mitochondrial NAD(P)+-dependent malic enzyme Ju-Yi Hsieh1,*, Yu-Hsiu Chiang1,*, Kuan-Yu Chang1 and Hui-Chih Hung1,2 Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan Institute of Bioinformatics, National Chung-Hsing University, Taichung, Taiwan Keywords allosteric regulation; analytical ultracentrifugation; electrostatic interaction; malic enzyme; mutagenesis Correspondence H.-C Hung, Department of Life Sciences and Institute of Bioinformatics, National Chung-Hsing University, 250, Kuo-Kuang Road, Taichung, 40227 Taiwan Fax: +886 22851856 Tel: +886 22840416 (ext 615) E-mail: hchung@dragon.nchu.edu.tw *These authors contributed equally to this work (Received 30 July 2008, revised 19 November 2008, accepted December 2008) doi:10.1111/j.1742-4658.2008.06834.x Here we report on the role of Glu59 in the fumarate-mediated allosteric regulation of the human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) In the present study, Glu59 was substituted by Asp, Gln or Leu Our kinetic data strongly indicated that the charge properties of this residue significantly affect the allosteric activation of the enzyme The E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold in enzyme activation with elevated activation constants, KA,Fum and aKA,Fum The E59D enzyme, although retaining the allosteric property, is quite different from the wild-type in enzyme activation The KA,Fum and aKA,Fum of E59D are also much greater than those of the wild-type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding Analytical ultracentrifugation analysis shows that both the wild-type and E59Q enzymes exist as a dimer–tetramer equilibrium In contrast to the E59Q mutant, the E59D mutant displays predominantly a dimer form, indicating that the quaternary stability in the dimer interface is changed by shortening one carbon side chain of Glu59 to Asp59 The E59L enzyme also shows a dimer–tetramer model similar to that of the wild-type, but it displays more dimers as well as monomers and polymers Malate cooperativity is not significantly notable in the E59 mutant enzymes, suggesting that the cooperativity might be related to the molecular geometry of the fumarate-binding site Glu59 can precisely maintain the geometric specificity for the substrate cooperativity According to the sequence alignment analysis and our experimental data, we suggest that charge effect and geometric specificity are both critical factors in enzyme regulation Glu59 discriminates human m-NAD-ME from mitochondrial NADP+-dependent malic enzyme and cytosolic NADP+-dependent malic enzyme in fumarate activation and malate cooperativity Malic enzyme (ME) comprises a family of oxidative decarboxylases that catalyze the transformation of the substrate l-malate to CO2 and pyruvate, with instantaneous reduction of NAD(P)+ to NAD(P)H [1–3] Divalent metal ions (Mn2+ or Mg2+) are essential for this enzymatic reaction These enzymes are universally present in nature, with conserved sequences, and have generally similar structural topology among different species [4–8] According to their cofactor specificity, mammalian ME has been divided into three isoforms: Abbreviations c-NADP-ME, cytosolic NADP+-dependent malic enzyme; ES, enzyme–substrate; ME, malic enzyme; m-NAD-ME, mitochondrial NAD(P)+dependent malic enzyme; m-NADP-ME, mitochondrial NADP+-dependent malic enzyme; PFK-1, phosphofructokinase-1 FEBS Journal 276 (2009) 983–994 ª 2009 The Authors Journal compilation ª 2009 FEBS 983 Human mitochondrial NAD(P)+-dependent malic enzyme J.-Y Hsieh et al cytosolic NADP+-dependent ME (c-NADP-ME) [9,10], mitochondrial NADP+-dependent ME (m-NADPME) [11], and mitochondrial NAD(P)+-dependent ME (m-NAD-ME) [1,11] Mitochondrial NAD(P)+-dependent ME has dual cofactor specificity, and can use both NAD+ and NADP+ as cofactor, but physiologically it favors NAD+ in maximizing its enzyme activity [1,12] Human m-NAD-ME may associated with the growth of highly proliferating tissues and tumors through the NADH and pyruvate produced in glutaminolysis [1,13–20] Unlike the other two mammalian isoforms, m-NAD-ME is a regulatory enzyme with a complex control system for manipulating its catalytic activity [21–23] The enzyme exhibits positive cooperative behavior with respect to the substrate l-malate, and it is an allosteric enzyme activated by fumarate [18,21– 27] Previous studies have suggested that ATP may act as an allosteric inhibitor of m-NAD-ME [18,21,25], and the allosteric properties of this isoform may relate to its particular role in the pathways of malate and glutamine oxidation in tumor mitochondria [17–21,24] However, further site-directed mutagenesis and kinetics studies showed that ATP may actually act as an active site inhibitor, rather than an allosteric inhibitor [28,29] The crystal structures of MEs demonstrate that the enzyme is a homotetramer with a double-dimer quaternary structure On the basis of structural information, MEs are categorized into a new class of oxidative decarboxylases with a novel backbone structure [4,6,8,30] The structures of human m-NAD-ME with malate ⁄ pyruvate, Mn2+ ⁄ Mg2+, NAD+, fumarate and transition state analog inhibitors have been resolved [4,5,31–33] In the structure of human m-NAD-ME, besides the active site, there are two regulatory sites (Fig 1A) One of them, located at the tetramer interface and called the exo site, is occupied by an NAD or ATP molecule The other is at the dimer interface, and is occupied by fumarate [32] The structures of pigeon c-NADP-ME and Ascaris suum m-NAD-ME in complex with various ligands have also been reported [6,7,30] These structures not show the additional exo site, but a separate allosteric site is found in the A suum m-NAD-ME at the dimer interface [26,27] Figure 1B shows the binding mode of fumarate at the dimer interface Structural studies have revealed that Arg67 and Arg91 are the ligands for fumarate binding The side chains of Arg91 and Arg67 form salt bridges with the carboxylate group of fumarate (Fig 1B) Site-directed mutagenesis and kinetic studies confirmed that Arg67 and Arg91 are indeed essential for fumarate activation Both R67S 984 and R91T mutant enzymes are insensitive towards fumarate [32] However, both Arg67 and Arg91 are conserved among other ME isoforms that are not activated by fumarate (Fig 1C) Thus, additional factors must be involved in governing the activation mechanism of fumarate in the human and A suum m-NAD-ME [27,32] In our previous work, we delineated the functional role of Asp102, which is close to the Arg67–fumarate– Arg91 ion pair network but does not directly interact with fumarate and is not conserved in other nonallosteric MEs (Fig 1C) We proposed that Asp102 is important for preserving the electrostatic balance in the fumarate-binding pocket, which may be a central factor in the regulatory mechanism of fumarate Mutation of Asp102 to Ala and Lys, however, abolishes the allosteric activation of the enzyme [23] In this article, we aimed to explore in detail the factors governing the allosteric regulation of the enzyme Previous studies have already shown that Glu59, a structural neighbor of Arg67, plays an important role in the fumarate activation of the enzyme Besides the R67S and R91T mutant enzymes, which are insensitive towards fumarate, the E59L mutant enzyme does not show any enzyme activation with fumarate, indicating that Glu59 has remarkable effects on allosteric activation [32] In the present study, the functional roles of Glu59 in the regulatory mechanism of the enzyme are elucidated Glu59 is substituted by Asp, Gln and Leu Detailed kinetic and analytical ultracentrifugation analyses of these mutants help to determine the factors affecting fumarate activation, subunit–subunit interaction and substrate cooperativity of human m-NAD-ME Results Kinetic parameters of the human wild-type and E59 mutant m-NAD-MEs The kinetic parameters of the wild-type and E59 mutant enzymes were determined with and without 20 mm fumarate (Table 1) Without fumarate, there were no significant differences in Km,NAD and K0.5,Malate observed among the wild-type and E59 mutant enzymes, except for the E59D enzyme The Km,NAD and K0.5,Malate values of E59D were 2-fold and 4.4-fold higher, respectively, than that of the wild-type Furthermore, the kcat value of E59 mutant enzymes was only one-third to one-quarter of that of the wild-type, suggesting that mutation of Glu59 in the fumarate-binding site causes the enzyme to become less efficient in catalysis FEBS Journal 276 (2009) 983–994 ª 2009 The Authors Journal compilation ª 2009 FEBS Human mitochondrial NAD(P)+-dependent malic enzyme J.-Y Hsieh et al A B C D Fig Fumarate-binding site for human m-NAD-ME (A) Homotetramer of human m-NAD-ME (Proein Data Bank code: 1PJ3) (B) Fumaratebinding site of m-NAD-ME The corresponding amino acids in the fumarate-binding site, Arg67, Arg91 and Glu59, are represented as a balland-stick model The color is yellow for fumarate This figure was generated with PYMOL (DeLano Scientific LLC, San Carlos, CA, USA) (C) Multiple sequence alignments of three clusters of ME isoforms around the fumarate-binding region are shown Amino acid sequences of MEs were obtained by a similarity search of BLAST [44], and alignments were created with CLUSTAL W [45] This figure was generated using the BIOEDIT sequence alignment editor program [46] (D) Glu59-binding ligands in the wild-type enzyme are shown as the LIGPLOT diagram [47] The bold bonds indicate the specific amino acid, the thin bonds are the hydrogen-bonded residues, and the green dashed lines correspond to the hydrogen bonds Spoked arcs represent hydrophobic contacts FEBS Journal 276 (2009) 983–994 ª 2009 The Authors Journal compilation ª 2009 FEBS 985 Human mitochondrial NAD(P)+-dependent malic enzyme J.-Y Hsieh et al Km,NAD (mM) Wild-type ) 0.88 + 0.43 E59Q ) 0.92 + 0.82 E59D ) 1.60 + 0.76 E59L ) 0.76 + 0.49 K0.5,Malate (mM) kcat (s)1) h ± 0.10 ± 0.04 15.44 ± 0.97 3.28 ± 0.37 208 ± 10.6 332 ± 15.3 1.84 ± 0.21 1.01 ± 0.09 ± 0.10 ± 0.06 15.23 ± 2.10 7.01 ± 1.13 74 ± 7.5 141 ± 10.3 1.24 ± 0.13 1.21 ± 0.07 ± 0.16 ± 0.04 67.22 ± 7.76 6.43 ± 0.68 57 ± 3.5 196 ± 8.6 1.03 ± 0.05 1.01 ± 0.11 ± 0.06 ± 0.04 10.68 ± 1.03 10.75 ± 1.91 53 ± 3.1 50 ± 4.1 1.20 ± 0.14 1.00 ± 0.08 With fumarate, the Km,NAD and K0.5,Malate of the wild-type enzyme decreased, the kcat values of the enzyme increased, and the h value, which represents the cooperativity of malate binding, was significantly reduced from 1.8 to 1, indicating the characteristics of allosteric regulation of the enzyme isoform by fumarate The E59Q enzyme, similar to the wild-type, showed a decrease in Km,NAD and K0.5,Malate but an increase in kcat However, a considerable decrease in malate cooperativity was observed in this mutant The h value was 1.2 with or without fumarate For the E59D enzyme, the Km,NAD and K0.5,Malate values decreased by about two-fold and 10-fold, respectively, and the kcat value of the mutant enzyme increased by three-fold The malate cooperativity, however, completely disappeared in this mutant The h value was 1.0, with or without fumarate Indeed, the E59D enzyme had an unusually large K0.5,Malate value, which could be reduced to a level similar to that of the wildtype by the addition of fumarate, suggesting that the active site of this mutant had been changed and was readjusted by fumarate For the E59L enzyme, the K0.5,Malate and kcat values were not notably influenced by fumarate, indicating that these mutant enzymes were insensitive to fumarate activation Activating effect of fumarate on the human wild-type and E59 mutant m-NAD-MEs The initial rates of m-NAD-ME measured in various concentrations of fumarate showed hyperbolic kinetics (Fig 2) At a saturating concentration of fumarate, the maximal activation by fumarate for the wild-type enzyme was approximately 1.5-fold, with an apparent KA value of 0.21 ± 0.03 mm (Fig 2, closed circles) 986 100 Velocity (µM·min–1) Table Kinetic parameters for the human wild-type and E59 mutant m-NAD-MEs ), no fumarate added; +, with 20 mM fumarate added 80 60 40 20 0 10 20 30 [Fumarate] (mM) 40 Fig Fumarate activation of the human wild-type and E59 mutant m-NAD-MEs The assay mixture contained ME (1.5 lg), 40 mM malate, 10 mM MgCl2, and mM NAD+, with various fumarate concentrations as indicated Closed circles, wild-type enzyme; closed triangles, E59Q enzyme; open circles, E59D enzyme; open triangles, E59L enzyme The maximal activation fold of the E59Q enzyme was similar to that of the wild-type enzyme, whereas the relative enzyme activity of the E59Q enzyme was only one-third of that of the wild-type (Fig 2, closed triangles), and it had a much higher apparent KA value (14.1 ± 2.3 mm) than that of the wild-type, suggesting that the E59Q enzyme needed more fumarate molecules to achieve its maximal activation The E59D enzyme could be activated more than three-fold, with an apparent KA value of 6.1 ± 1.1 mm, a value that was also much higher than that of the wild-type (Fig 2, open circles) Even though the maximal activation fold of the E59D enzyme was more than that of the wild-type, the relative enzyme activity of the E59D enzyme was raised to the level of one-half of that of the wild-type (Fig 2, open circles) Like the E59Q enzyme, the E59D enzyme also required more fumarate to achieve this activation The E59L enzyme could not be activated by fumarate (Fig 2, open triangles) These preliminary results indicate that the side chain properties of residue 59 seem to have a great impact on the allosteric regulation of ME Activation constants of the human wild-type and E59 mutant m-NAD-MEs The activation constants of the wild-type and E59 mutant enzymes were further determined by kinetic analysis The enzyme activities were assayed in a broad range of substrate malate concentrations at different fixed concentrations of fumarate (data not shown) These curves were globally fitted to Eqn (1), and the activation constant of fumarate for free enzyme (KA,Fum) and for the enzyme–substrate (ES) complex (aKA,Fum) were estimated FEBS Journal 276 (2009) 983–994 ª 2009 The Authors Journal compilation ª 2009 FEBS Human mitochondrial NAD(P)+-dependent malic enzyme J.-Y Hsieh et al Table Activation constants of fumarate and dissociation constants of dimer–tetramer equilibrium for the human wild-type and E59 mutant m-NAD-MEs KA,Fum (mM) Wild-type E59Q E59D E59L aKA,Fum (mM) Kd (lM) 0.73 ± 0.11 13.1 ± 3.9 8.5 ± 1.8 – 0.17 ± 0.03 5.63 ± 1.68 3.91 ± 0.83 – 6.27 7.93 4395 10.21 ± ± ± ± DG (kcalỈmol)1) 0.05 0.06 23 0.10 )7.0 )6.8 )3.2 )6.7 ± ± ± ± 0.05 0.05 0.02 0.07 The activation parameters of the wild-type and E59 mutant human enzymes are summarized in Table The values of KA,Fum and aKA,Fum with respect to malate for the wild-type enzyme were 0.73 mm and 0.17 mm, respectively, indicating that fumarate binds the ES complex more tightly than free enzyme For the E59Q enzyme, the values of KA,Fum and aKA,Fum were 13.1 and 5.63 mm, respectively, greater than those of the wild-type by at least an order of magnitude (18fold and 33-fold, respectively), indicating that the binding affinity of fumarate for the E59Q enzyme was markedly less than that of the wild-type The negative charge of Glu59 is important for the binding affinity of fumarate to either free enzyme or the ES complex For the E59D enzyme, the KA,Fum and aKA,Fum values were 8.5 and 3.91 mm, respectively, also larger than those of the wild-type enzyme by over 10-fold This reveals that although the negative charge was conserved, the activation constants of fumarate for the E59D enzyme were considerably elevated, reflecting a decreased binding affinity of fumarate Self-association of the human wild-type and E59 mutant m-NAD-MEs The fumarate-binding site resides at the dimer interface (Fig 1A) We use analytical ultracentrifugation to examine the possible change in the quaternary structure of E59 mutant enzymes Figure shows the continuous sedimentation coefficient distribution of the wild-type and E59 mutants The sedimentation coefficients of 6.5 S and 9.0 S represented the dimer and tetramer, respectively, corresponding to molecular masses of 124 and 248 kDa The quaternary structure of the wild-type is in dimer–tetramer equilibrium with different protein concentrations (Fig 3A–C) The E59Q enzyme apparently shows a similar dimer– tetramer pattern as the wild-type (Fig 3D–F), both being in dimer form in low protein concentrations and being reconstituted into tetramers in high protein concentrations In contrast to the E59Q enzyme, the E59D enzyme has a predominantly dimer form, with a few monomers and polymers in the range of protein concentrations used (Fig 3G–I), indicating that the quaternary stability in the dimer interface is greatly changed by shortening one carbon side chain of Glu59 to Asp59 The E59L enzyme also showed a dimer– tetramer model similar to that of the wild-type, but it displayed more dimers as well as monomers and polymers, which are not observed to a significant extent in the wild-type WT (Fig 3J–L) In order to estimate the self-association of enzymes quantitatively, the sedimentation velocity data were analyzed globally to determine the dissociation constant (Kd) of the wild-type and E59 mutants (Fig 4) Structural data show that the interactions in the tetramer interface are weaker than those in the dimer interface (Fig 1A); thus, the Kd value of the wild-type may reflect the dissociation between the A and D or B and C subunits to form AB or CD dimers The wild-type and E59Q enzymes had similar Kd values of 6.3 and 7.9 lm, respectively (Table 2), showing that the subunit–subunit interactions in the dimer interface were not disrupted by substituting a negatively charged Glu with a neutrally charged Gln The E59D enzyme, although conserving the negative charge on residue 59, still altered its dimer–tetramer equilibrium into a dominant dimer form with a Kd value of 4395 lm, which is over 600-fold larger than that of the wild-type As Glu59 is near the dimer interface, the E59D dimer might be an AD (BC) dimer If this is the case, the Kd value of the E59D enzyme may represent the dissociation in the dimer interface The E59L enzyme is also in dimer– tetramer equilibrium, with a slightly larger Kd value than the wild-type, suggesting that the Leu substitution did not cause substantial changes in the dimer interface In the presence of fumarate, the dimer–tetramer equilibrium of the wild-type was shifted (Fig 5A) With the addition of fumarate, most dimeric enzymes reconstitute into tetrameric enzymes, suggesting that fumarate stabilizes the tetrameric state of the enzyme, which may increase the catalytic activity Similar to the wild-type, the E59Q enzyme was also changed in its dimer–tetramer equilibrium by fumarate (Fig 5B), suggesting that the tetramer organization was not perturbed in this mutant The E59D enzyme, however, could not be reconstituted into a tetramer by fumarate (Fig 5C); it existed in a dimeric form in the presence of fumarate Although the quaternary structure of the E59L enzyme displayed a model similar to that of the wild-type (Fig 3), the dimer–tetramer equilibrium of this mutant enzyme could not be shifted by fumarate (Fig 5D), suggesting that the mutant enzyme is insensitive to fumarate with regard not only to catalytic activity but also to quaternary structure organization FEBS Journal 276 (2009) 983–994 ª 2009 The Authors Journal compilation ª 2009 FEBS 987 Human mitochondrial NAD(P)+-dependent malic enzyme J.-Y Hsieh et al Fig Continuous sedimentation coefficient distribution of the human wild-type and E59 mutant m-NAD-MEs The enzymes were used at three protein concentrations, 0.2, 0.6 and 1.2 mgỈmL)1 in 50 mM Tris ⁄ HCl buffer (pH 7.4) at 20 °C (A–C) Wild-type (D–F) E59Q (G–I) E59D (J–L) E59L Discussion Binding network of the allosteric activator fumarate in human m-NAD-ME Fumarate has been identified as the allosteric activator for human m-NAD-ME by decreasing the Km values of the active site ligands [13,21,23,28] As well as the 988 mammal enzymes, m-NAD-ME from A suum is also activated by fumarate [27] As the crystal structure of human m-NAD-ME in complex with fumarate has been determined, the binding network of fumarate in the enzyme has become clear In the fumarate-binding pocket, two arginyl residues, Arg67 and Arg91, have been identified as determining the major binding affinity of fumarate for the enzyme An anionic amino FEBS Journal 276 (2009) 983–994 ª 2009 The Authors Journal compilation ª 2009 FEBS Human mitochondrial NAD(P)+-dependent malic enzyme J.-Y Hsieh et al Fig Global analysis of sedimentation velocity data of the human wild-type and E59 mutant m-NAD-MEs at three protein concentrations Sedimentation was performed at 20 °C with an An-50 Ti rotor and at a rotor speed of 42 000 r.p.m (A–C) Concentrations of the protein were 0.2, 0.6 and 1.2 mgỈmL)1, respectively The symbols are raw sedimentation data, and the lines are data fitted by the software SEDPHAT (D–F) The fitting residuals of the model from the upper panel afford a reliable analysis result for a dissociation constant (Kd) of the monomer–dimer equilibrium Fig Continuous sedimentation coefficient distribution of the human wild-type and E59 mutant m-NAD-MEs in the presence of fumarate The enzyme concentration was fixed at 0.2 mgỈmL)1 without (solid lines) or with (broken lines) mM (broken lines) or mM (dotted lines) fumarate (A) Wild-type (B) E59Q (C) E59D (D) E59L acid, Glu59, which forms salt bridges with Arg67 in the structure but is not found in nonallosteric MEs, is suggested to be important for fumarate activation [32] In this study, we examined the effect of the charge and hydrogen bonding network of the Glu59 side chain on allosteric regulation of the enzyme Charge effect of residue 59 on enzyme regulation In the structure of the wild-type enzyme, Glu59 is ionpaired and hydrogen-bonded with Arg67 and Lys57 (Fig 1D) Simultaneously, fumarate is coordinated with Arg67 and Arg91 in the structure; the ionic pairs FEBS Journal 276 (2009) 983–994 ª 2009 The Authors Journal compilation ª 2009 FEBS 989 Human mitochondrial NAD(P)+-dependent malic enzyme J.-Y Hsieh et al and hydrogen bonding network among Glu59, Arg67, fumarate and Arg91 may be responsible for fumarateinduced allosteric regulation (Fig 1B) Our kinetic data strongly indicated that the charge properties of Glu59 significantly affect the allosteric activation of the enzyme (Fig 2) The fact that the E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold for enzyme activation clearly indicates that the charge effect is an influential factor in fumarate activation Abolishing the salt bridges between Glu59 and Arg67 has a significant effect on the binding network of fumarate The E59Q enzyme can still be activated by fumarate, and the maximal activation fold is similar to that of the wild-type, whereas achieving the maximal activation requires a higher fumarate concentration Significant differences in KA,Fum and aKA,Fum were observed for the E59Q enzyme, suggesting that the negative charge of Glu has a significant impact on the binding affinity of fumarate As the molecular dimensions and polarity of Gln are close to those of Glu, the proper conformational geometry and hydrogen bonding network should be preserved even though the negative charge of Glu is replaced by a neutral side chain of Gln Thus, the delayed activation of this mutant enzyme could be attributed to the ion pairs derived from Glu59 being destroyed by this replacement For the E59D enzyme, despite the negative charge of the residue and the allosteric property of the enzyme being retained, the kinetic properties are quite different from those of the wild-type The KA,Fum and aKA,Fum values of the E59D enzyme are also much greater than those of the wild-type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding Furthermore, kinetic analysis demonstrates that the E59D enzyme resembles a partial inactive enzyme with an anomalously high Km value and low enzyme activity in the absence of fumarate This may result from shortening the length of the side chain in the enzyme, causing geometrical changes in the fumarate-binding pocket, which may have an effect on the active site The structural change in the E59D enzyme may be adjusted by binding of fumarate, and the mutant enzyme can thus be reactivated Subunit–subunit interaction in the dimer interface and malate cooperativity Analytical ultracentrifugation analysis demonstrates that the wild-type enzyme exists in dimer–tetramer equilibrium in solution The quaternary structure of the E59Q enzyme is as stable as that of the wild-type, 990 demonstrating a similar dimer–tetramer pattern (Figs and 5) and a similar dissociation constant (Table 2) This fact suggests that the molecular geometry in the dimer interface is not significantly changed by the substitution of Gln Unlike the E59Q enzyme, the E59D enzyme exists mainly as a dimer rather than in dimer–tetramer equilibrium (Fig 3) The dissociation of subunits in the dimer interface might be caused by the structural change in the E59D enzyme The ionic interactions between Asp59 and Lys57 might still occur, but be somewhat altered because of the shorter side chain of Asp59 In the wildtype, Lys57 is located in the dimer interface; its side chain is hydrogen-bonded not only with Glu59 but also with Pro216 and Tyr218 from the other subunit Analytical ultracentrifugation analysis shows that mutation of Lys57 causes the enzyme to dissociate into unstable dimers and, further, to form polymers (J.-Y Hsieh, Y.W Fang & H.-C Hung, unpublished results) In the E59D enzyme, Lys57 might be pulled by Asp59 into one subunit, thus being no longer hydrogen-bonded with Pro216 and Tyr218 from the other subunit, and finally leading to the disintegration of tetramers of the enzyme This can be fitted well with the explanation of why the E59L enzyme is kept in dimer–tetramer equilibrium It can be concluded that the hydrophobic Leu introduced did not influence the subunit interactions of Lys57 However, the monomer and polymer appearing in the equilibrium may have been caused by the environmental alteration from hydrophilic to hydrophobic Although Glu59 is near the dimer interface, it is also possible that the E59D dimers are AB (and CD) dimers, based on the fact that the tetramer is a dimer of AB (and CD) dimers Another possibility could be that the E59D enzyme maintains a disrupted AB dimer, and the conformational disturbance somehow prevents the formation of the tetramer However, this question can be definitively answered until the biophysical data for discrimination between AB and AD dimers are available The malate cooperativity in the E59 mutant enzymes is almost abolished This is not surprising in the E59D enzyme, because this mutant enzyme is mainly in dimer form For the E59Q and E59L enzymes, even though the former conserves the allosteric activation of fumarate, and like the wild-type, both principally reserve a dimer–tetramer equilibrium, their malate cooperativity still decreases significantly The loss of cooperativity might be related to the alteration in molecular geometry of the fumarate-binding site Our data suggest that only Glu59 can precisely hold the geometric specificity of the allosteric site and that this is important for substrate cooperativity FEBS Journal 276 (2009) 983–994 ª 2009 The Authors Journal compilation ª 2009 FEBS Human mitochondrial NAD(P)+-dependent malic enzyme J.-Y Hsieh et al Involvement of the allosteric triad in the regulatory mechanism of human m-NAD-ME Structural data have clearly revealed that both Arg67 and Arg91 are the direct ligands for fumarate It is not surprising that mutation of these argininyl residues causes the enzyme to become insensitive to fumarate [32] According to the sequence alignments, neither Arg67 nor Arg91 are specifically conserved in m-NAD ME, the only isoform that could be activated by fumarate In fact, Arg91 is highly conserved among all classes of MEs, whereas Arg67, although moderately conserved, is completely conserved among all mammalian ME isoforms (Fig 1E) Glu59, however, is conserved only in m-NAD-ME (Fig 1C); in m-NADPME and c-NADP-ME, this residue is replaced by Leu or Asn, respectively Hence, the latter two enzymes are not fumarate-activated In fact, the E59L and E59Q enzymes display, as such, the properties of a nonregulatory ME The E59L m-NAD-ME is insensitive to fumarate, showing nonallosteric and noncooperative kinetics, whereas E59Q m-NAD-ME displays a much higher activation threshold and less cooperativity Glu59 thus discriminates human m-NAD-ME from m-NADP-ME and c-NADP-ME in fumarate regulation and malate cooperativity Glu59, Arg67 and Arg91 form an allosteric triad in the fumarate site of human m-NAD-ME The allosteric triad is the basic element for fumarate binding, and thus determines whether the enzyme is allosteric or nonallosteric Many enzymes in the metabolic pathway are controlled by its allosteric regulator Besides human m-NAD-ME, many enzymes in the metabolic pathway are allosteric enzymes The one that is most well characterized is phosphofructokinase1 (PFK-1) [34] PFK-1 catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate Fructose 2,6-bisphosphate is the potent allosteric activator for this regulatory enzyme [35,36] In the absence of fructose 2,6-bisphosphate, the enzyme is almost inactive at the physiological concentrations of its substrate, fructose 6-phosphate When fructose 2,6-bisphosphate binds to the allosteric site on PFK-1, it increases the substrate affinity, with a significant decrease of K0.5 from mm to 0.08 mm Similar to the effect of fumarate on human m-NAD-ME, fructose 2,6-bisphosphate activates PFK-1 by increasing the apparent affinity for fructose 6-phosphate The regulatory mechanism of these two enzymes gives a good example of the reactivation–deactivation of an alloste- ric enzyme controlled by its specific allosteric regulator produced in the metabolic pathway Experimental procedures Expression and purification of recombinant MEs The detailed expression and purification protocols for human m-NAD-ME have been reported in earlier studies [1,31] In brief, m-NAD-ME was subcloned into the expression vector (pRH281) and transformed into Escherichia coli BL21 cells for enzyme overexpression by controlling the inducible trp promoter system [1] Anionic exchange, DEAE–Sepharose (Amersham Biosciences, Uppsala, Sweden), followed by ATP–agarose affinity chromatography (Sigma, St Louis, MO, USA) were employed in the enzyme purification The purified enzyme was subsequently bufferexchanged and concentrated in 30 mm Tris ⁄ HCl (pH 7.4) and mm b-mercaptoethanol by a centrifugal filter device (Amicon Ultra-15; Millipore, Billerica, MA, USA) with a molecular mass cutoff of 30 kDa The enzyme purity was checked by SDS ⁄ PAGE, and the protein concentrations were estimated by the Bradford method [37] Site-directed mutagenesis Site-directed mutagenesis was carried out using the QuikChange kit (Stratagene, La Jolla, CA, USA) The purified DNA of human m-NAD-ME was used as a template, and the primers with the desired codons were employed to change Glu59 into Asp, Gln and Leu, using a high fidelity of Pfu DNA polymerase in the PCR reaction Primers including the mutation site are 25- to 45-mer, which is considered necessary for specific binding of template DNA The synthetic oligonucleotides used in these site-directed mutagenesis experiments were 5¢-GGACTTCTACCTCCCAAAATA GACACACAAGATATTCAAGCC-3¢ for E59D, 5¢-GGAC TTCTACCTCCCAAAATACAGACACAAGATATTCAA GCC-3¢ for E59Q, and 5¢-GGACTTCTACCTCCCAAAA TACTGACACAAGATATTCAAGCC-3¢ for E59L The nucleotides underlined and marked in bold indicate the mutation positions After 16–18 temperature cycles, the mutated plasmids including staggered nicks were made The PCR products were subsequently treated with DpnI to digest the wild-type human m-NAD-ME templates Finally, the nicked DNA with desired mutations was transformed into E coli strain XL-1, and their DNA sequences were checked by autosequencing Enzyme kinetic analysis Enzymatic activity of MEs was measured by the reduction of NAD+ to NADH The reaction mixture contained FEBS Journal 276 (2009) 983–994 ª 2009 The Authors Journal compilation ª 2009 FEBS 991 Human mitochondrial NAD(P)+-dependent malic enzyme J.-Y Hsieh et al 50 mm Tris ⁄ HCl (pH 7.4), 40 mm malate (pH 7.4), 2.0 mm NAD+ and 10 mm MgCl2 in a total volume of mL The absorbance at 340 nm at 30 °C was instantaneously traced after the enzyme was added to the reaction mixture, and monitored continuously in a Beckman DU 7500 spectrophotometer Under these conditions, unit of the enzyme was defined as the amount of enzyme catalyzing the production of lmol of NADH per An extinction coefficient of 6.22 per mm for NADH was utilized in the calculations Apparent Michaelis constants of the substrate and cofactors were determined by varying the concentration of one substrate (or cofactor) around its Km value, while keeping other components constant at the saturating concentrations The nonessential activation model was employed to estimate the dissociation constants for free enzyme (E) and ES complex [38] The experiment was carried out at a series of fumarate concentrations and at different concentrations of l-malate The total set of data was globally fitted to the following equation, which was derived from a nonessential activation mechanism (Scheme 1) [38]: m=Vmax ẳ ẵS=fKs ỵ ẵA=KA;Fum ị=1 ỵ bẵA=aKA;Fum ị ỵ ẵS ỵ ẵA=KA;Fum ị=1 ỵ bẵA=aKA;Fum Þg ð1Þ in which v is the observed initial velocity, and Vmax is the maximum rate of the unactivated reaction The maximum rate in the presence of fumarate is bVmax Ks is the Michaelis constant for the substrate, and KA,Fum and aKA,Fum are the activation constants for fumarate binding to free enzyme (E) and ES complex, respectively The sigmoidal curves of [malate] versus initial rates were fitted into the Hill equation, and data were further analyzed to calculate the K0.5 value, the substrate concentration at half-maximal velocity, and the Hill coefficient (h), which were employed to assess the degree of cooperativity Quaternary structure analysis by analytical ultracentrifugation Sedimentation velocity experiments were carried out using a Beckman Optima XL-A analytical ultracentrifuge Sample (380 lL) and buffer (400 lL) solutions were loaded into the double sector centerpiece separately, and built up in a Beckman An-50 Ti rotor Experiments were performed at 20 °C and a rotor speed of 42 000 r.p.m Protein samples were monitored by UV absorbance at 280 nm in a continuous mode with a time interval of 480 s and a step size of 0.002 cm Multiple scans at different time points were fitted to a continuous size distribution model by the program sedfit [39–42] All size distributions were solved at a confidence level of P = 0.95, a best-fitted average anhydrous frictional ratio (f ⁄ f0), and a resolution N of 200 sedimentation coefficients between 0.1 and 20.0 S To precisely determine the dissociation constants of MEs in dimer–tetramer equilibrium, sedimentation velocity experiments were performed with three different protein concentrations of the enzyme All sedimentation data were globally fitted to the monomer–dimer equilibrium model of the program sedphat to calculate the dissociation constant (Kd) of the enzyme [41] The partial specific volume of the enzyme, solvent density and viscosity were calculated by the software program sednterp [43] Acknowledgements This work was supported by the National Science Council, ROC (NSC-96-2311-B-005-005 to H.-C Hung), and in part by the Ministry of Education, Taiwan, ROC under the ATU plan We thank Professor G G Chang (Faculty of Life Sciences and the Institute of Biochemistry, National Yang-Ming University) for critically reading the manuscript h m ẳ Vmax ẵmalateh =K0:5 ỵ ẵmalateh ị References All data-fitting work was carried out with the sigma plot 8.0 program (Jandel, San Rafael, CA, USA) E+S Ks kP ES E+P + + A (Fum) A (Fum) KA,Fum EA + S KA,Fum Ks ESA kP EA + P Scheme Nonessential activation mechanism of human mitochondrial NAD(P)+-dependent malic enzyme 992 Loeber G, Infante AA, Maurer-Fogy I, Krystek E & Dworkin MB (1991) Human NAD+-dependent mitochondrial malic enzyme J Biol Chem 266, 3016– 3021 Cleland WW (2000) Chemical mechanism of malic enzyme as 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LIGPLOT: a program to generate schematic diagrams of protein–ligand interactions Protein Eng 8, 127–134 FEBS Journal 276 (2009) 983–994 ª 2009 The Authors Journal compilation ª 2009 FEBS ... Fumaratebinding site of m-NAD-ME The corresponding amino acids in the fumarate- binding site, Arg67, Arg91 and Glu59, are represented as a balland-stick model The color is yellow for fumarate. .. active site The structural change in the E59D enzyme may be adjusted by binding of fumarate, and the mutant enzyme can thus be reactivated Subunit–subunit interaction in the dimer interface and. .. in the fumarate site of human m-NAD-ME The allosteric triad is the basic element for fumarate binding, and thus determines whether the enzyme is allosteric or nonallosteric Many enzymes in the

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