Biology 403, Spring 2004, Note-Sheet for Third Midterm

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Biology 403, spring 2004, note-sheet for third midterm Energy associated with protonmotive force: ΔG = ΔGchem + ΔGelec = -2.303nRT(pHin - pHout) + zFΔψ Protonmotive force: Δp = ΔG/(nF) = Δψ - 2.303RTΔpH / F Glycolysis, Pyruvate Dehydrogenase, and Citric Acid Cycle: Reactants Products Enzyme Glucose,ATP G-6-P, ADP, H+ hexokinase G-6-P F-6-P Glucose-6-phosphate isomerase F-6-P,ATP F-1,6-diP,ADP,H+ phosphofructokinase-1 F-1,6-bisP DHAP, Glycald-3-P aldolase DHAP Glycald-3-P Triosephosphate Isomerase + + Glycald-3-P, NAD ,Pi 1,3-bis-P-glycerate,NADH,H Glycald-3P dehydrogenase 1,3-bis-P-glycerate,ADP 3-P-glycerate,ATP phosphoglycerate kinase 3-P-glycerate 2-P-glycerate phosphoglycerate mutase 2-P-glycerate PEP + H2O enolase PEP,ADP,H+ pyruvate, ATP pyruvate kinase + + Pyruvate, HS-CoA, NAD CO2,NADH,H ,Acetyl CoA pyruvate dehydrogenase acetyl CoA,oxalacetate,H2O citrate citrate synthase citrate isocitrate aconitase isocitrate,NAD+ α-k-glutarate,NADH,CO2 isoictrate dehydrogenase α-k-glutarate, HS-CoA,NAD+ succinyl CoA,NADH,CO2 α-k-glutarate dehydrogenase succinyl CoA,GDP,Pi succinate,GTP,HS-CoA succinyl CoA synthase Succinate,Q fumarate,QH2 succinate dehydrogenase fumarate,H2O L-malate fumarate hydratase L-malate,NAD+ oxaloacetate,NADH,H+ malate dehydrogenase Reactions unique to gluconeogenesis: Reactants Products Enzyme - Pyruvate, HCO3 ,ATP oxaloacetate+ADP+Pi pyruvate carboxylase oxaloacetate,GTP PEP+GDP+CO2 PEP carboxykinase F-1,6-P F-6-P+Pi F-1,6-bisphosphatase G-6-P glucose + Pi glucose 6-phosphatase Redox Complexes in the Electron Transport Chain: Complex ΔEo', V ΔGo', kJ/mol Complex ΔEo', V ΔGo', kJ/mol I (NADH/Q) +0.36 -70 II (Succinate/Q) +0.01 -2 III (QH2/cyto C) +0.19 -37 IV (Cyto C/O2 +0.59 -110 Lipoproteins: Property Chylomicrons VLDLs IDLs LDLs HDLs MW*10-6 >400 10-80 5-10 2.3 0.18-0.36 Density, g cm-3 <0.95 0.95-1.006 1.006-1.019 1.019-1.063 1.063-1.210 % Protein 2 10 18 25 33 % Triacylglycerol 85 50 31 10 8 % Cholesterol 4 22 29 45 30 % Phospholipid 9 18 22 20 29 Beta Oxidation of Fatty Acids: Reactants Products Enzyme 2 Fatty acyl CoA, FAD trans-Δ -Enoyl CoA, FADH2 Acyl-CoA dehydrogenase 3+ 2+ FADH2,Fe-S FAD,Fe-S Electron-transferring flavoprotein 2+ 3+ Fe-S ,Q Fe-S , QH2 ETF:ubiquinone reductase 2 trans-Δ -Enoyl CoA,H2O L-3-hydroxyacyl CoA Enoyl-CoA hydratase L-3-hydroxyacyl CoA, NAD+ 3-ketoacyl CoA,NADH,H+ L-3-Hydroxyacyl-CoA dehydrogenase acetyl CoA, 3-ketoacyl CoA,HS-CoA thiolase shortened fatty acyl CoA Comparison of fatty acid oxidation and synthesis in mammals: Property oxidation synthesis localization mitochondria, peroxisomes cytosol acyl carrier CoA acyl carrier protein carbon units C2 C2 acceptor / donor CoA (C2) malonyl CoA (evolves CO2) mobile redox cofactors NAD+, ETF, Q NADPH enzyme organization separate enzymes multifunctional enzyme.

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Construction and Enzymatic Characterization of E
1 Minimizing acetate formation in E. coli fermentations 2 Marjan De Mey*, Sofie De Maeseneire, Wim Soetaert and Erick Vandamme 3 Ghent University, Laboratory of Industrial Microbiology and Biocatalysis, Department of 4 Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Coupure links 5 653, B-9000 Ghent, Belgium 6 7 * Correspondence address: Marjan De Mey 8 Laboratory of Industrial Microbiology and Biocatalysis 9 Department of Biochemical and Microbial Technology 10 Faculty of Bioscience Engineering 11 Ghent University 12 Coupure links 653 13 B-9000 Gent 14 Tel: +32-9-264-60-28 15 Fax: +32-9-264-62-31 16 [email protected] 17 Keywords: acetate reduction, Escherichia coli, metabolic engineering 1 18 Abstract 19 Escherichia coli remains the best established production organisms in industrial 20 biotechnology. However, during aerobic fermentation runs at high growth rates, considerable 21 amounts of acetate are accumulated as by-product. This by-product has negative effects on 22 growth and protein production. Over the last 20 years, substantial research efforts have been 23 spent to reduce acetate accumulation during aerobic growth of E. coli on glucose. From the 24 onset it was clear that this quest should not be a simple nor uncomplicated one. Simple 25 deletion of the acetate pathway, reduced the acetate accumulation, but instead other by- 26 products were formed. This minireview gives a clear outline of these research efforts and the 27 outcome of them, including bioprocess level approaches and genetic approaches. Recently, 28 the latter seems to have some promising results. 29 30 1 Introduction 31 Escherichia coli was the first and is still one of the most commonly used production 32 organisms in industrial biotechnology.
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Targeting Pyruvate Carboxylase Reduces Gluconeogenesis and Adiposity and Improves Insulin Resistance Naoki Kumashiro,1,2 Sara A
ORIGINAL ARTICLE Targeting Pyruvate Carboxylase Reduces Gluconeogenesis and Adiposity and Improves Insulin Resistance Naoki Kumashiro,1,2 Sara A. Beddow,3 Daniel F. Vatner,2 Sachin K. Majumdar,2 Jennifer L. Cantley,1,2 Fitsum Guebre-Egziabher,2 Ioana Fat,2 Blas Guigni,2 Michael J. Jurczak,2 Andreas L. Birkenfeld,2 Mario Kahn,2 Bryce K. Perler,2 Michelle A. Puchowicz,4 Vara Prasad Manchem,5 Sanjay Bhanot,5 Christopher D. Still,6 Glenn S. Gerhard,6 Kitt Falk Petersen,2 Gary W. Cline,2 Gerald I. Shulman,1,2,7 and Varman T. Samuel2,3 – We measured the mRNA and protein expression of the key transcriptional factors and cofactors (8 12). Yet, despite gluconeogenic enzymes in human liver biopsy specimens and the high degree of transcription regulation for these found that only hepatic pyruvate carboxylase protein levels enzymes, the control they exert over gluconeogenic ﬂux is related strongly with glycemia. We assessed the role of pyruvate relatively weak (13–16). We recently reported that hepatic carboxylase in regulating glucose and lipid metabolism in rats expression of PEPCK and G6PC mRNA was not related to through a loss-of-function approach using a speciﬁc antisense fasting hyperglycemia in two rodent models of type 2 di- oligonucleotide (ASO) to decrease expression predominantly in abetes and in patients with type 2 diabetes (17). Thus, we liver and adipose tissue. Pyruvate carboxylase ASO reduced hypothesized that other mechanisms must account for in- plasma glucose concentrations and the rate of endogenous glucose production in vivo. Interestingly, pyruvate carboxylase ASO also creased hepatic gluconeogenesis and fasting hyperglycemia reduced adiposity, plasma lipid concentrations, and hepatic in type 2 diabetes.
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Citric Acid Cycle
CHEM464 / Medh, J.D. The Citric Acid Cycle Citric Acid Cycle: Central Role in Catabolism • Stage II of catabolism involves the conversion of carbohydrates, fats and aminoacids into acetylCoA • In aerobic organisms, citric acid cycle makes up the final stage of catabolism when acetyl CoA is completely oxidized to CO2. • Also called Krebs cycle or tricarboxylic acid (TCA) cycle. • It is a central integrative pathway that harvests chemical energy from biological fuel in the form of electrons in NADH and FADH2 (oxidation is loss of electrons). • NADH and FADH2 transfer electrons via the electron transport chain to final electron acceptor, O2, to form H2O. Entry of Pyruvate into the TCA cycle • Pyruvate is formed in the cytosol as a product of glycolysis • For entry into the TCA cycle, it has to be converted to Acetyl CoA. • Oxidation of pyruvate to acetyl CoA is catalyzed by the pyruvate dehydrogenase complex in the mitochondria • Mitochondria consist of inner and outer membranes and the matrix • Enzymes of the PDH complex and the TCA cycle (except succinate dehydrogenase) are in the matrix • Pyruvate translocase is an antiporter present in the inner mitochondrial membrane that allows entry of a molecule of pyruvate in exchange for a hydroxide ion. 1 CHEM464 / Medh, J.D. The Citric Acid Cycle The Pyruvate Dehydrogenase (PDH) complex • The PDH complex consists of 3 enzymes. They are: pyruvate dehydrogenase (E1), Dihydrolipoyl transacetylase (E2) and dihydrolipoyl dehydrogenase (E3). • It has 5 cofactors: CoASH, NAD+, lipoamide, TPP and FAD. CoASH and NAD+ participate stoichiometrically in the reaction, the other 3 cofactors have catalytic functions.
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Mechanism F Formation of Oxaloacetate and Phosphoenol Pyruvate from Pyruvate1
THE JOURNAL OF VITAMINOLOGY 14, 59-67 (1968) MECHANISM F FORMATION OF OXALOACETATE AND PHOSPHOENOL PYRUVATE FROM PYRUVATE1 HARLAND G. WOOD Department of Biochemistry, Case Western Reserve University Cleveland, Ohio 44, 106, U.S.A. Pyruvate, oxaloacetate and P-enolpyruvate have been known to be key compounds in metabolism for many years; pyruvate since 1911 when Neuberg found that yeast decarboxylates pyruvate to acetaldehyde, oxaloacetate since 1937 when Krebs pro posed the citric acid cycle, and P-enolpyruvate since 1934-1936 when Lohmann, Meyerhof and Kiessling discovered that 3-phosphoglycerate is converted to P enolpyruvate by dialyzed yeast or muscle extracts. These three compounds are the hub of numerous reactions leading to both synthesis and breakdown of carbo hydrates, fats and nitrogenous compounds (See the recent review by Utter (1) ). Furthermore, they are interlocked within themselves by a series of reactions re sulting in the synthesis of each fromn the other with accompanying breakdown of the companion compound. There has been a long debate concerning the mechanism of formation of P enolpyruvate from pyruvate. The conversion of P-enolpyruvate to pyruvate in glycolysis by pyruvate kinase was shown by Lardy and Ziegler (2) in 1945 to be reversible, but the equilibrium lies far to the right and the physiological signi ficance of the reversibility is questionable. The possibility that P-enolpyruvate might be formed by a pathway other than via pyruvate kinase was first raised by Kalckar (3) who found that kidney preparations could form P-enolpyruvate from malate and fumarate. At that time Wood and Werkman had discovered fixation of CO2 by heterotrophs and had postulated that the fixation occurs by combination of CO2 with pyruvate to give oxaloacetate.
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(12) United States Patent (10) Patent No.: US 7927,859 B2 San Et Al
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