Mechanism of malic enzyme from pigeon liver. Magnetic resonance and kinetic studies of the role of Mn2+.

Hsu, Robert Y.; Mildvan, Albert S.; Chang, Gu‐Gang; Fung, Chien H.

doi:10.1016/s0021-9258(17)32986-1

Cited by 72 publications

(46 citation statements)

References 32 publications

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“…Lower concentrations of Mn2+ also provided less but substantial protection (Figure 3A). The [Mn2+] giving 50% protection was ~7.5 /iM, in excellent agreement to the £d value of the tight Mn2+-binding site of the enzyme (6-10 jiM) (Hsu et al, 1976). Mg2+, Zn2+, Co2+, or Ca2+ at 4 mM also afforded 100% protection.…”

Section: Inactivation Of Pigeon Liver Malic Enzyme By Ferroussupporting

confidence: 74%

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Affinity Cleavage at the Putative Metal-Binding Site of Pigeon Liver Malic Enzyme by the Fe2+-Ascorbate System

Wei¹,

Chou²,

Huang³

et al. 1994

Biochemistry

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Pigeon liver malic enzyme was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at neutral pH and 0 or 25 degrees C. Omitting the ascorbate or replacing the ferrous ion with manganese ion did not lead to any inactivation. Manganese, magnesium, zinc, cobalt, or calcium ion at 200 molar excess over ferrous ion offered complete protection of the enzyme from Fe(2+)-induced inactivation. Ni2+ provided partial protection, while Ba2+ or imidazole was ineffective in protection. Addition of 4 mM Mn2+ or 5 mM EDTA into a partially modified enzyme stopped further inactivation of the enzyme. Inclusion of substrates (L-malate or NADP+, singly or in combination) in the incubation mixture did not affect the inactivation rate. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native pigeon liver malic enzyme had a subunit M(r) of 65,000. The inactivated enzyme with residual activity of only 0.3% was cleaved into two fragments with M(r) of 31,000 and 34,000, respectively. The cleavage site was identified as the peptide bond between Asp258 and Ile259. Native pigeon liver malic enzyme was blocked at the N-terminus. Cleavage at the putative metal-binding site exposed a new N-terminus, which was identified to be at the 34-kDa fragment containing the C-terminal half of original sequence 259-557. Our results indicated that Fe2+ catalyzed a specific oxidation of pigeon liver malic enzyme at Asp258 and/or some other essential amino acid residues that caused enzyme inactivation. The modified enzyme was then affinity cleaved at the Mn(2+)-binding site.

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Section: Inactivation Of Pigeon Liver Malic Enzyme By Ferroussupporting

confidence: 74%

“…Malic enzyme requires divalent metal cation for catalysis. The function of metal ion is to polarize the carbonyl groups, forming a second-sphere complex with the substrate (Hsu et al, 1976). Thus, metal ion plays a central role in transition-state stabilization for the malic enzyme-catalyzed reaction.…”

Section: Discussionmentioning

confidence: 99%

Affinity Cleavage at the Putative Metal-Binding Site of Pigeon Liver Malic Enzyme by the Fe2+-Ascorbate System

Wei¹,

Chou²,

Huang³

et al. 1994

Biochemistry

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“…To explain these phenomena explicitly, we propose a working model for the quaternary structure of these recombinant malic enzymes as shown in Figure 3. The wild-type malic enzyme is presented as an asymmetric double dimer to account for the various kinetic and dissociation observations (A in Figure 3) (Hsu et al, 1976;Chang & Hsu, 1977a;Dalziel et al, 1983;Chang et al, 1988Chang et al, , 1993Chang et al, , 1994Lee & Chang, 1990;Huang & Chang, 1992). We propose that Phe 19 is located in a region which connects malic enzyme and the maltose binding protein.…”

Section: Mutation Of Malic Enzymementioning

confidence: 77%

Involvement of Phe¹⁹ in the Mn²⁺−l-Malate Binding and the Subunit Interactions of Pigeon Liver Malic Enzyme

et al. 1996

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A triple mutant, F19S/N250S/L353Q, of pigeon liver malic enzyme was found to have no detectable enzymatic activity [Chou, W.-Y., Huang, S.-M., & Chang, G.-G. (1994) Arch. Biochem. Biophys. 310, 158-166]. In the present study, point mutants at these positions (F19S, N250S, and L353Q) were prepared by site-directed mutagenesis. Both N250S and L353Q have kinetic properties similar to those of the wild-type. On the other hand, the K(m)(app) values for both Mn2+ and L-malate of F19S were increased by approximately 10-fold, while the kcat value was decreased by 5-fold, which results in a decrease of the apparent catalytic efficiency (kcat/K(mNADP)K(mMal)K(mMn) by approximately 300-fold. These results clearly indicate that the F19S mutation is mainly responsible for the undetectable enzyme activity of the triple mutant. Three more Phe19 mutants (F19Y, F19G, and F19A) were then prepared. There is a direct correlation between the size of the substitutes and the affinities for Mn2+ and L-malate. The kinetic parameters for F19Y were similar to those for wild-type. Both F19A and F19G reveal a 5-fold decrease of kcat values. Two K(dMn) values for the high- and low-affinity sites, respectively, were detectable for the wild-type. On the contrary, only one K(dMn) value was detected for the F19 mutants, which was increased in the order of F19G > F19A > F19S > F19Y, with F19G being the most affected mutant. The K(mMal) values of F19G and F19A were increased 100- and 6-fold, respectively. The catalytic efficiency (kcat/K(mNADP)K(dMal)K(dMn)) of F19G was decreased to only 0.01% of that of the wild-type. The above results clearly indicate that the hydrophobic aromatic ring at position 19 plays a critical role in L-malate and Mn2+ binding. Furthermore, all mutants that have a small residue at position 19 exist as monomers. Therefore, Phe19 may locate in or near the regions for Mn(2+)-L-malate binding as well as for the subunit contact. These results are compatible with the asymmetric model for the quaternary structure of malic enzyme we proposed previously [Chang, G.-G., Huang, T.-M., Huang, S.-M., & Chou, W.-Y. (1994) Eur. J. Biochem. 225, 1021-1027]. The possible roles of the N-terminus of malic enzyme were also addressed.

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“…In the functionally related enzyme ICDH, which also has a two step catalytic mechanism, one of the aspartate ligands of the cation was proposed as the general base and a tyrosine or lysine residue was proposed as the general acid [56]. The situation with ME is more complicated, as a water-mediated interaction between the cation and the substrate is expected [4,58]. This might increase the distance between the aspartate/glutamate ligands of the cation and malate, making them less likely to function as the general base.…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases

Bhargava

et al. 1999

Structure

144

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Background: Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate and CO(2) with the concomitant reduction of NAD(P)(+) to NAD(P)H. They are widely distributed in nature and have important biological functions. Human mitochondrial NAD(P)(+)-dependent malic enzyme (mNAD-ME) may have a crucial role in the metabolism of glutamine for energy production in rapidly dividing cells and tumors. Moreover, this isoform is unique among malic enzymes in that it is a cooperative enzyme, and its activity is controlled allosterically. Results: The crystal structure of human mNAD-ME has been determined at 2.5 Å resolution by the selenomethionyl multiwavelength anomalous diffraction method and refined to 2.1 Å resolution. The structure of the monomer can be divided into four domains; the active site of the enzyme is located in a deep cleft at the interface between three of the domains. Three acidic residues (Glu255, Asp256 and Asp279) were identified as ligands for the divalent cation that is required for catalysis by malic enzymes. Conclusions: The structure reveals that malic enzymes belong to a new class of oxidative decarboxylases. The tetramer of the enzyme appears to be a dimer of dimers. The active site of each monomer is located far from the tetramer interface. The structure also shows the binding of a second NAD(+) molecule in a pocket 35 Å away from the active site. The natural ligand for this second binding site may be ATP, an allosteric inhibitor of the enzyme.

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Mechanism of malic enzyme from pigeon liver. Magnetic resonance and kinetic studies of the role of Mn2+.

Cited by 72 publications

References 32 publications

Affinity Cleavage at the Putative Metal-Binding Site of Pigeon Liver Malic Enzyme by the Fe2+-Ascorbate System

Affinity Cleavage at the Putative Metal-Binding Site of Pigeon Liver Malic Enzyme by the Fe2+-Ascorbate System

Involvement of Phe¹⁹ in the Mn²⁺−l-Malate Binding and the Subunit Interactions of Pigeon Liver Malic Enzyme

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases

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Mechanism of malic enzyme from pigeon liver. Magnetic resonance and kinetic studies of the role of Mn2+.

Cited by 72 publications

References 32 publications

Affinity Cleavage at the Putative Metal-Binding Site of Pigeon Liver Malic Enzyme by the Fe2+-Ascorbate System

Affinity Cleavage at the Putative Metal-Binding Site of Pigeon Liver Malic Enzyme by the Fe2+-Ascorbate System

Involvement of Phe19 in the Mn2+−l-Malate Binding and the Subunit Interactions of Pigeon Liver Malic Enzyme

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases

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Involvement of Phe¹⁹ in the Mn²⁺−l-Malate Binding and the Subunit Interactions of Pigeon Liver Malic Enzyme