Reduction of alpha-oxo carboxylic acids by pigeon liver 'malic' enzyme.
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ABSTRACT:
SUBMITTER: Tang CL
PROVIDER: S-EPMC1165821 | biostudies-other | 1973 Oct
REPOSITORIES: biostudies-other
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Project description:The pH-induced reversible dissociation of pigeon liver malic enzyme (EC 1.1.1.40) was studied by combined use of chemical cross-linking and SDS/polyacrylamide-gel electrophoresis. The tetrameric enzyme showed a pH-dependent dissociation in an acidic environment. At pH values above 8.0 most molecules existed as tetramers. The enzyme was gradually dissociated at lower pH. When the pH was below 5.0 most of the enzyme was present as the monomeric forms. Reassociation of the subunits was accomplished by adjusting the pH to neutrality. The dissociation and reassociation were almost instantaneous. No trimer was detected. The pigeon liver malic enzyme was thus shown to have a double-dimer quaternary structure with D2 symmetry. In the presence of substrates, the monomer-dimer-tetramer equilibrium favours the direction of dissociation. Tartronate, an L-malate analogue, was found to be more effective than L-malate in this process. When the monomeric forms were immobilized, the enzyme subunits were found to be fully active in catalysis. A possible arrangement of the four identical subunits of the enzyme molecule is proposed to account for the results obtained in this investigation. The origin of the half-of-the-sites reactivity of pigeon liver malic enzyme is also discussed.
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Project description:The copper hydride (CuH)-catalyzed enantioselective reduction of α,β-unsaturated carboxylic acids to saturated aldehydes is reported. This protocol provides a new method to access a variety of β-chiral aldehydes in good yields, with high levels of enantioselectivity and broad functional group tolerance. A reaction pathway involving a ketene intermediate is proposed based on preliminary mechanistic studies and density functional theory calculations.
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Project description:Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO(2) in the presence of divalent cations (Mg(2+), Mn(2+)). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD(+) or NADP(+), but not both. Structural studies of the human mitochondrial NAD(+)-dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP(+)-dependent malic enzyme, in a closed form, in a quaternary complex with NADP(+), Mn(2+), and oxalate. This represents the first structural information on an NADP(+)-dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2'-phosphate group of the NADP(+) cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP(+) selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.
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Project description:Cytosolic malic enzyme (Me1) was predicted as a causal gene for abdominal using a novel statistical method named LCMS (Schadt et al., 2005, Nature Genetics). In order to validate this prediction, we profiled the liver tissues of Me1 knockout mice (Me1-/-) and their littermate wild-type (wt) controls to examine the gene expression signature as well as pathways/networks resulting from the single gene perturbation.
2009-03-08 | GSE11992 | GEO
Project description:Cytosolic malic enzyme (Me1) was predicted as a causal gene for abdominal using a novel statistical method named LCMS (Schadt et al., 2005, Nature Genetics). In order to validate this prediction, we profiled the liver tissues of Me1 knockout mice (Me1-/-) and their littermate wild-type (wt) controls to examine the gene expression signature as well as pathways/networks resulting from the single gene perturbation. 3 Me1-/- mice and 5 wt controls were profiled. Reference pool included RNA extracted from the liver of 5 wt control mice. Dye-swap was involved in the profiling.
2009-03-07 | E-GEOD-11992 | biostudies-arrayexpress
Project description:The cytosolic malic enzyme from pigeon liver is very sensitive to the metal-catalysed oxidation systems. Our previous studies using the Cu2+-ascorbate as the oxidation system showed that the enzyme was oxidized and cleaved at several positions, including Asp141. The recently resolved crystal structure of pigeon liver malic enzyme revealed that Asp141 was near to the metal-binding site, but was not a direct metal ligand. However, Asp141 is located next to Phe236, which directly follows the metal ligands Glu234 and Asp235. Mutation at Asp141 caused a drastic effect on the metal-binding affinity of the enzyme. Since Asp141 and Phe236 are highly conserved in most species of malic enzyme, we used a double-mutant cycle to study the possible interactions between these two residues. Four single mutants [D141A (Asp141-->Ala), D141N, F236A and F236L] and four double mutants (D141A/F236A, D141N/F236A, D141A/F236L and D141N/F236L), plus the wild-type enzyme were successfully cloned, expressed and purified to homogeneity. The secondary, tertiary and quaternary structures of these mutants, as assessed by CD, fluorescence and analytical ultracentrifuge techniques, were similar to that of the wild-type enzyme. Initial velocity experiments were performed to derive the various kinetic parameters, which were used to analyse further the free energy change and the coupling energy (DeltaDeltaG(int)) between any two residues. The dissociation constants for Mn2+ ( K (d,Mn)) of the D141A and F236A mutants were increased by approx. 6- and 65-fold respectively, compared with that of the wild-type enzyme. However, the K (d,Mn) for the double mutant D141A/F236A was only increased by 150-fold. A coupling energy of -2.12 kcal/mol was obtained for Asp141 and Phe236. We suggest that Asp141 is involved in the second sphere of the metal-binding network of the enzyme.
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