- Identification of the active site residues in ATP-citrate lyase's carboxy-terminal portion
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ATP-citrate lyase (ACLY) catalyzes production of acetyl-CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)-terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C-terminal portion of ACLY, full-length C. limicola ACLY was cleaved, first non-specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C-terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.
- Nguyen, Vinh H.,Singh, Noreen,Medina, Ana,Usón, Isabel,Fraser, Marie E.
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- Increasing the conformational entropy of the Ω-loop lid domain in phosphoenolpyruvate carboxykinase impairs catalysis and decreases catalytic fidelity
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Many studies have shown that the dynamic motions of individual protein segments can play an important role in enzyme function. Recent structural studies of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) demonstrate that PEPCK contains a 10-residue Ω-loop domain that acts as an active site lid. On the basis of these structural studies, we have previously proposed a model for the mechanism of PEPCK catalysis in which the conformation of this mobile lid domain is energetically coupled to ligand binding, resulting in the closed conformation of the lid, necessary for correct substrate positioning, becoming more energetically favorable as ligands associate with the enzyme. Here we test this model by introducing a point mutation (A467G) into the center of the Ω-loop lid that is designed to increase the entropic penalty for lid closure. Structural and kinetic characterization of this mutant enzyme demonstrates that the mutation has decreased the favorability of the enzyme adapting the closed lid conformation. As a consequence of this shift in the equilibrium defining the conformation of the active site lid, the enzymes ability to stabilize the reaction intermediate is weakened, resulting in catalytic defect. This stabilization is initially surprising, as the lid domain makes no direct contacts with the enolate intermediate formed during the reaction. Furthermore, during the conversion of OAA to PEP, the destabilization of the lid-closed conformation results in the reaction becoming decoupled as the enolate intermediate is protonated rather than phosphorylated, resulting in the formation of pyruvate. Taken together, the structural and kinetic characterization of A467G-PEPCK supports our model of the role of the active site lid in catalytic function and demonstrates that the shift in the lowest-energy conformation between open and closed lid states is a function of the free energy available to the enzyme through ligand binding and the entropic penalty for ordering of the 10-residue Ω-loop lid domain.
- Johnson, Troy A.,Holyoak, Todd
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- An investigation of the equilibrium of the reaction {l-aspartate(aq) + 2-oxoglutarate(aq) = oxaloacetate(aq) + l-glutamate(aq)}
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Apparent equilibrium constants have been measured for the following biochemical reaction: L-aspartate(aq) + 2-oxoglutarate(aq) = oxaloacetate(aq) + L-glutamate(aq). This reaction, catalysed by aspartate transaminase, was studied over the ranges 283.15 ≤ T/K ≤ 303.15, 6.94 ≤ pH ≤ 7.13, and 0.163 ≤ Im/(mol·kg-1) ≤ 0.167, where T is temperature and Im is ionic strength. The instability of the oxaloacetate in solution required the use of an experimental procedure that was brief. Thus, the procedure used was to measure the change in the chromatographic response ΔR of the oxaloacetate chromatographic peak that accompanied the reaction. Values of ΔR were measured for several solutions under near equilibrium conditions. The chromatographic response ΔR is expected to be zero for a solution that is at equilibrium with regard to the above reaction and prior to the addition of the enzyme. The results were used to calculate the standard molar Gibbs energy change ΔrGom = (4.82 ± 0.21) kJ·mol-1, the equilibrium constant K = (0.143 ± 0.012), the standard molar enthalpy change ΔrHom = (1.9 ± 2.9) kJ±mol-1, and the standard molar entropy change ΔrSom = -(10 ± 10) J·K-1·mol-1 for the following chemical reference reaction at T = 298.15 K and Im = 0: L-aspartate-(aq) + 2-oxoglutarate2-(aq) = oxaloacetate2-(aq) + L-glutamate-(aq). Under near physiological conditions (T = 311.15 K, pH = 7.0, Im = 0.25 mol·kg-1) the apparent equilibrium constant K′ for the overall biochemical reaction is calculated to have the value 0.147; the standard transformed Gibbs energy change ΔrG′om = 4.96 kJ·mol-1 under these conditions.
- Kishore, Nand,Tewari, Yadu B.,Goldberg, Robert N.
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- Structural and kinetic characterization of 4-hydroxy-4-methyl-2- oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate aldolase, a protocatechuate degradation enzyme evolutionarily convergent with the HpaI and DmpG pyruvate aldolases
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4-Hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate (HMG/CHA) aldolase from Pseudomonas putida F1 catalyzes the last step of the bacterial protocatechuate 4,5-cleavage pathway. The preferred substrates of the enzyme are 2-keto-4-hydroxy acids with a 4-carboxylate substitution. The enzyme also exhibits oxaloacetate decarboxylation and pyruvate α-proton exchange activity. Sodium oxalate is a competitive inhibitor of the aldolase reaction. The pH dependence of kcat/Km and kcat for the enzyme is consistent with a single deprotonation with pKa values of 8.0 ± 0.1 and 7.0 ± 0.1 for free enzyme and enzyme substrate complex, respectively. The 1.8 A x-ray structure shows a four-layered α-β-β-α sandwich structure with the active site at the interface of two adjacent subunits of a hexamer; this fold resembles the RNase E inhibitor, RraA, but is novel for an aldolase. The catalytic site contains a magnesium ion ligated by Asp-124 as well as three water molecules bound by Asp-102 and Glu-199′. A pyruvate molecule binds the magnesium ion through both carboxylate and keto oxygen atoms, completing the octahedral geometry. The carbonyl oxygen also forms hydrogen bonds with the guanadinium group of Arg-123, which site-directed mutagenesis confirms is essential for catalysis. A mechanism for HMG/CHA aldolase is proposed on the basis of the structure, kinetics, and previously established features of other aldolase mechanisms.
- Wang, Weijun,Mazurkewich, Scott,Kimber, Matthew S.,Seah, Stephen Y. K.
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- Role of cysteine residues in 4-oxalomesaconate hydratase from pseudomonas ochraceae NGJ1
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4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wildand mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the Km-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl2; and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.
- Li, Suhong,Kimura, Maho,Takashima, Teruo,Hayashi, Kunihiko,Inoue, Kazunori,Ishiguro, Ryou,Sugisaki, Hiroyuki,Maruyama, Kiyofumi
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- Dehydration and Enolization Rates of Oxalacetate: Catalysis by Tertiary Amines
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The hydration/dehydration and tautomerization rates of oxalacetate are subject to acid and base catalysis.H2O and OH- are much more effective for the former process than for the latter, but the opposite holds for tertiary amine catalysis.In buffers comprised of oxyanion bases or unsaturated amines, dehydration is significantly faster than tautomerization and remains so as the buffer concentration is varied.In dilute tertiary amine buffers dehydration rates are also faster, but as the buffer concentration increases, tautomerization rates increase rapidly and eventually surpass those for hydration/dehydration.It is this rate crossover which led earlier workers (ref 9) to propose a carbinolamine mechanism for a tertiary amine-catalyzed tautomerization.After the rate crossover is taken into account, enolization is found to show linear rate-buffer concentration behavior with tertiary amines, vitiating the prime evidence cited for the carbinolamine mechanism.However, tertiary amine catalysis likely operates differently in the tautomerization and dehydration reactions.
- Emly, Mark,Leussing, Daniel L.
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- Mechanistic Studies on Tryptophan Lyase (NosL): Identification of Cyanide as a Reaction Product
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Tryptophan lyase (NosL) catalyzes the formation of 3-methylindole-2-carboxylic acid and 3-methylindole from l-tryptophan. In this paper, we provide evidence supporting a formate radical intermediate and demonstrate that cyanide is a byproduct of the NosL-catalyzed reaction with l-tryptophan. These experiments require a major revision of the NosL mechanism and uncover an unanticipated connection between NosL and HydG, the radical SAM enzyme that forms cyanide and carbon monoxide from tyrosine during the biosynthesis of the metallo-cluster of the [Fe-Fe] hydrogenase.
- Bhandari, Dhananjay M.,Fedoseyenko, Dmytro,Begley, Tadhg P.
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supporting information
p. 542 - 545
(2018/01/26)
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- Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase
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Pyridoxal 5'-phosphate (PLP) is a fundamental, multifunctional enzyme cofactor used to catalyze a wide variety of chemical reactions involved in amino acid metabolism. PLP-dependent enzymes optimize specific chemical reactions by modulating the electronic states of PLP through distinct active site environments. In aspartate aminotransferase (AAT), an extended hydrogen bond network is coupled to the pyridinyl nitrogen of the PLP, influencing the electrophilicity of the cofactor. This network, which involves residues Asp-222, His-143, Thr-139, His-189, and structural waters, is located at the edge of PLP opposite the reactive Schiff base. We demonstrate that this hydrogen bond network directly influences the protonation state of the pyridine nitrogen of PLP, which affects the rates of catalysis. We analyzed perturbations caused by single-and double-mutant variants using steady-state kinetics, high resolution X-ray crystallography, and quantum chemical calculations. Protonation of the pyridinyl nitrogen to form a pyridinium cation induces electronic delocalization in the PLP, which correlates with the enhancement in catalytic rate in AAT. Thus, PLP activation is controlled by the proximity of the pyridinyl nitrogen to the hydrogen bond microenvironment. Quantum chemical calculations indicate that Asp-222, which is directly coupled to the pyridinyl nitrogen, increases the pKa of the pyridine nitrogen and stabilizes the pyridinium cation. His-143 and His-189 also increase the pKa of the pyridine nitrogen but, more significantly, influence the position of the proton that resides between Asp-222 and the pyridinyl nitrogen. These findings indicate that the second shell residues directly enhance the rate of catalysis in AAT.
- Dajnowicz, Steven,Parks, Jerry M.,Hu, Xiche,Gesler, Korie,Kovalevsky, Andrey Y.,Mueser, Timothy C.
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p. 5970 - 5980
(2017/04/17)
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- Identification of fumarate hydratase inhibitors with nutrient-dependent cytotoxicity
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Development of cell-permeable small molecules that target enzymes involved in energy metabolism remains important yet challenging. We describe here the discovery of a new class of compounds with a nutrient-dependent cytotoxicity profile that arises from p
- Takeuchi, Toshifumi,Schumacker, Paul T.,Kozmin, Sergey A.
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supporting information
p. 564 - 567
(2015/01/30)
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- Caged CO2 for the Direct Observation of CO2-Consuming Reactions
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CO2-consuming reactions, in particular carboxylations, play important roles in technical processes and in nature. Their kinetic behavior and the reaction mechanisms of carboxylating enzymes are difficult to study because CO2 is inconvenient to handle as a gas, exists in equilibrium with bicarbonate in aqueous solution, and typically yields products that show no significant spectroscopic differences from the reactants in the UV/Vis range. Here we demonstrate the utility of 3-nitrophenylacetic acid and related compounds (caged CO2) in conjunction with infrared spectroscopy as widely applicable tools for the investigation of such reactions, permitting convenient measurement of the kinetics of CO2 consumption. The use of isotopically labeled caged CO2 provides a tool for the assignment of infrared absorption bands, thus aiding insight into reaction intermediates and mechanisms.
- Lommel, Katharina,Sch?fer, Gabriela,Grenader, Konstantin,Ruland, Christoph,Terfort, Andreas,M?ntele, Werner,Wille, Georg
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p. 372 - 380
(2013/08/24)
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- Kinetic mechanism and structural requirements of the amine-catalyzed decarboxylation of oxaloacetic acid
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(Chemical Equation Presented) The kinetic and chemical mechanism of amine-catalyzed decarboxylation of oxaloacetic acid at pH 8.0 has been reevaluated using a new and versatile assay. Amine-catalyzed decarboxylation of oxaloacetic acid proceeds via the formation of an imine intermediate, followed by decarboxylation of the intermediate and hydrolysis to yield pyruvate. The decrease in oxaloacetic acid was coupled to NADH formation by malate dehydrogenase, which allowed the rates of both initial carbinolamine formation (as part of the imination step) and decarboxylation to be determined. By comparing the rates observed for a variety of amines and, in particular, diamines, the structural and electronic requirements for diamine-catalyzed decarboxylation at pH 8.0 were identified. At pH 8.0, monoamines were found to be very poor catalysts, whereas some diamines, most notably ethylenediamine, were excellent catalysts. The results indicate that the second amino group of diamines enhances the rate of imine formation by acting as a proton shuttle during the carbinolamine formation step, which enables diamines to overcome high levels of solvation that would otherwise inhibit carbinolamine, and thus imine, formation. The presence of the second amino group may also enhance the rate of the carbinolamine dehydration step. In contrast to the findings of previous reports, the second amino group participates in the reaction by enhancing the rate of decarboxylation via hydrogen-bonding to the imine nitrogen to either stabilize the negative charge that develops on the imine during decarboxylation or preferentially stabilize the reactive imine over the unreactive enamine tautomer. These results provide insight into the precise catalytic mechanism of several enzymes whose reactions are known to proceed via an imine intermediate.
- Thalji, Nabil K.,Crowe, William E.,Waldrop, Grover L.
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supporting information; experimental part
p. 144 - 152
(2009/04/07)
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