1	Pentose Pathway
2	The next 3 slides collectively make up the complete cycle.
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6	"6 HEXOSE-P" 6 HEXOSE-P + 6 O₂ 6 PENTOSE-P + 6 CO₂ + 6 H₂O 2 PENTOSE-P + 2 PENTOSE-P 2 TRIOSE-P + 2 HEPTOSE-P 2 TRIOSE-P + 2 HEPTOSE-P 2 HEXOSE-P + 2 TETROSE-P 2 TETROSE-P + 2 PENTOSE-P 2 HEXOSE-P + 2 TRIOSE-P 2 TRIOSE-P + 1 H₂O 1 HEXOSE-P + H₃PO₄ ________________________________________________________ HEXOSE-P + 6 O₂ 6 CO₂ + 5 H₂O + H₃PO₄
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8	"Reactions may act independently or..." Reactions may act independently or in concert with the glycolytic pathway. Not well defined path with end products, but a set of diverging pathways capable of great metabolic flexibility. Very common in microbial and plant tissue - less in mammals. 20 % of Glucose in rat liver proceeds via pentose pathway Heart and skeletal muscle - None Mammary gland requires NADP⁺, H⁺, hence lots of gluconate pathway
9	Krebs Cycle Citric Acid Cycle Tricarboxylic Acid Cycle
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14	Pyruvate dehydrogenase is link between Glycolysis and Krebs
15	Pyruvate Dehydrogenase Complex: structure of the thiamin diphosphate dependent enzyme pyruvate decarboxylase (the E₁ part of the complex) 1pyd.pdb)
16	Pyruvate Dehydrogenase Complex: The lipoyl E₂ domain of complex which serves as an acyltransferase. (1iyu.pdb)
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18	Step 1: Condensation: In step 1 of the Krebs cycle, the two-carbon compound, acetyl-S-CoA, participates in a condensation reaction with the four-carbon compound, oxaloacetate, to produce citrate:
19	"This reaction is moderately exergonic" This reaction is moderately exergonic. Thermodynamically, the equilibrium is in favor of the products. Thus, this is considered to be the first committed step of the Krebs cycle Being the first committed step, this is a likely step to have some kind of regulatory control mechanism (which will effectively regulate the entire cycle) The Krebs cycle is also known as the citric acid cycle. Citrate is a tricarboxylic acid, and the Krebs cycle is also known as the tricarboxylic acid (or TCA) cycle
20	Citrate Synthase: Citrate Synthase (E.C.4.1.3.7) complexed with oxaloacetate and amidocarboxy-methyldethia coenzyme A. (1csh.pdb)
21	Step 2. Isomerization of Citrate As we will see later on in the Krebs cycle, there will be a decarboxylation reaction. Such decarboxylation reactions usually involve b - (or a -) keto acids The hydroxyl group of citrate cannot be oxidized to yield a keto group (III alcohol) so to form a keto acid the hydroxyl must become secondary. Thus, step 2 involves moving the hydroxyl group in the citrate molecule so that we can later form an a-keto acid This process involves the fist mechanistic step of acid catalyzed dehydration to form a carbocation. Equilibrium between the II and III forms allows rearrangement to the less favored D-Isocitrate isomer (with the hydroxyl group now in the desired a- location). Cis-Aconitase is often shown but is not a required intermediate
22	A single enzyme, Aconitase, performs this two-step process:
23	There are two asymmetric centers in the D-Isocitrate molecule. Each can adopt either the R- or S- configuration, thus there are 4 possible isomers of this molecule
24	"Note:" Note: the stereospecificity of Aconitase was established by introducing carboxyl-labeled Acetate into the Krebs cycle. The conversion of Acetate into Acetyl-SCoA can subsequently result in the labeling of Citrate. Although Citrate is a symmetric molecule, the labeled carboxyl-group always ends up on the g- carbon group in S_D-Isocitrate
25	Aconitase: Aconitase (E.C.4.2.1.3) in the activated (4Fe-4S) cluster form. (6acn.pdb)
26	Step 3: Generation of CO₂ by an NAD⁺ linked enzyme The Krebs cycle contains two oxidative decarboxylation steps; this is the first one The reaction is catalyzed by the enzyme Isocitrate dehydrogenase
27	"The reaction involves dehydrogenation to..." The reaction involves dehydrogenation to Oxalosuccinate, an unstable intermediate which spontaneously decarboxylates to give a-Ketoglutarate The reaction is exergonic, with a DG^0' = -20.9 kJ/mol. This helps drive the preceding (endergonic) reaction in the cycle In addition to decarboxylation, this step produces a reduced nicotinamide adenine dinucleotide (NADH) cofactor, or a reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor If the NAD⁺ cofactor is reduced, then the D-Isocitrate must be oxidized when forming a-Ketoglutarate. Thus, this step is referred to as an oxidative decarboxylation step
28	Isocitrate Dehydrogenase: Isocitrate Dehydrogenase (E.C.1.1.1.42) with NADP⁺ (9icd.pdb)
29	Step 4: A Second Oxidative Decarboxylation Step The multi-step reaction performed by the a-Ketoglutarate Dehydration Complex is analogous to the Pyruvate Dehydrogenase Complex, i.e. an a-keto acid undergoes oxidative decarboxylation with formation of an acyl-SCoA Overall, this oxidative decarboxylation step is more exergonic than the first oxidative decarboxylation step
30	Step 5: Substrate-Level Phosphorylation
31	In plants and bacteria ATP is formed directly in the Succinyl-SCoA Synthase catalyzed reaction by phosphorylation of ADP directly. In animals, GDP is the substrate in the reaction with formation of GTP (which is then used to form ATP by Nucleoside Diphosphokinase)
32	Step 6: Flavin-Dependent Dehydrogenation The Succinate produced by Succinyl SCoA-Synthetase in the prior reaction needs to be converted to Oxaloacetate to complete the Krebs cycle. Both Succinate and Oxaloacetate are 4-carbon compounds The first step in the conversion is the dehydrogenation of Succinate to yield Fumarate
33	Succinyl-CoA Synthetase: Succinyl-CoA Synthetase (E.C.6.2.1.5) with coenzyme A (1scu.pdb)
34	"In this reaction a C-C..." In this reaction a C-C bond is being oxidized to produce a C=C bond. This oxidation is energetically more costly than oxidizing a C-O bond. The redox coenzyme for this reaction is therefore FAD, rather than NAD⁺. FAD is covalently bound to the Succinate Dehydrogenase molecule (via a histidine residue) The FADH₂ has to be oxidized for the enzyme activity to be restored. This oxidation occurs via interaction with the mitochondrial electron transport system (later). Succinate Dehydrogenase is tightly bound to the mitochondrial inner membrane Succinate Dehydrogenase is stereo-specific: the trans- isomer (Fumarate) is produced and not the cis- isomer (Maleate)
35	Step 7: Hydration of a Carbon-Carbon Double Bond
36	Fumarase: Fumarase with bound malate. (1fup.pdb)
37	Step 8: A Dehydrogenation Reaction that will Regenerate Oxaloacetate This is a highly endergonic reaction (DG^0' = +29.7 J/mol) and so the equilibrium strongly favors the reactants. However, the next step in the Krebs cycle is the highly exergonic reaction (DG^0' = -32.2 kJ/mol) catalyzed by Citrate Synthase and this keeps the levels of Oxaloacetate low (<10^-6 M) The formation of Oxaloacetate completes the Krebs cycle
38	Malate Dehydrogenase:Malate Dehydrogenase (E.C.1.1.1.37) a complex of the apoenzyme and citrate (2cmd.pdb)
39	"Stoichiometry and Energetics of the..." Stoichiometry and Energetics of the Citric Acid Cycle Reaction Enzyme DG^0' (kJ/mol) Acetyl-CoA + Oxaloacetate + H₂O ð Citrate + CoA-SH + H⁺Citrate Synthase -32.2 Citrateó cis-Aconitate + H₂O Aconitase +6.3 cis-Aconitase + H₂O ó Isocitrate Isocitrate + NAD⁺ ó a-Ketoglutarate + CO₂ + NADH Isocitrate Dehydrogenase -8.4 a-Ketoglutarate + NAD⁺ + CoA-SH ó Succinyl-CoA + CO₂ + NADH a-Ketoglutarate Dehydrogenase -33.5 Succinyl-CoA + Pi + GDP ó Succinate + GTP + CoA-SH Succinyl-CoA Synthetase -2.9 Succinate + E-FAD ó Fumarate + E-FADH₂Succinate Dehydrogenase 0 Fumarate + H₂O ó L-Malate Fumarase -3.8 L-Malate + NAD⁺ ó Oxaloacetate + NADH + H⁺Malate Dehydrogenase +29.7 NET:-44.8
40	"Acetyl-CoA + Oxaloacetate + H₂O..." Acetyl-CoA + Oxaloacetate + H₂O ð Citrate + CoA-SH + H⁺ Citrateó cis-Aconitate + H₂O cis-Aconitase + H₂O ó Isocitrate isocitrate + NAD⁺ ó a-Ketoglutarate + CO₂ + NADH a-Ketoglutarate + NAD⁺ + CoA-SH ó Succinyl-CoA + CO₂ + NADH Succinyl-CoA + Pi + GDP ó Succinate + GTP + CoA-SH Succinate + E-FAD ó Fumarate + E-FADH₂ Fumarate + H₂O ó L-Malate L-Malate + NAD⁺ ó Oxaloacetate + NADH + H⁺ Acetyl-CoA + 2H₂O + 3NAD⁺+ Pi + GDP + FAD ð 2CO₂ + 3NADH + GTP + CoA-SH + FADH₂ + 2H⁺
41	"One turn of the citric..." One turn of the citric acid cycle generates: One high-energy phosphate through substrate-level phosphorylation Three NADH One FADH₂
42	"Catabolism of Glucose through Glycolysis..." Catabolism of Glucose through Glycolysis and the Krebs Cycle Each molecule of Glucose produces two molecules of Pyruvate Glucose + 2NAD⁺ + 2ADP + 2Pi ð 2Pyruvate + 2NADH + 2H⁺ + 2H₂O +2ATP Action of Pyruvate Dehydrogenase on Pyruvate: Pyruvate + CoA-SH + NAD⁺ ð CO₂ + Acetyl-CoA + NADH The overall catabolism of Glucose to 2 Pyruvate molecules: Glucose + 2NAD⁺ + 2ADP + 2Pi ð 2Pyruvate + 2NADH + 2H⁺ + 2H₂O +2ATP 2Pyruvate + 2CoA-SH + 2NAD⁺ ð 2CO₂ + 2Acetyl-CoA + 2NADH Glucose + 4NAD⁺ + 2ADP + 2CoA-SH + 2Pi ð 2CO₂ + 2Acetyl- CoA + 4NADH + 2H⁺ + 2H₂O +2ATP The GTP formed in the animal Succinyl-CoA Synthetase reaction in the Krebs cycle is readily converted to ATP (by Nucleoside Diphosphokinase)
43	The PDB files on these pages were obtained from the Protein Data Bank, and were created by the authors listed. The Protein Data Bank, maintained by the Research Collaboratory for Structural Bioinformatics is an archival database of the three-dimensional structures of biological macromolecules such as proteins, nucleic acids and carbohydrates. The structures are derived from experimental work such as X-ray diffraction studies and NMR investigations. The contents of PDB are in the public domain, but it is expected that the authors of an entry as well as the PDB be properly cited whenever their work is referred. The following is the current citation for the PDB: H.M.Berman, J.Westbrook, Z.Feng, G.Gilliland, T.N.Bhat, H.Weissig, I.N.Shindyalov, P.E.Bourne "The Protein Data Bank" Nucleic Acids Research 2000, 28, 235-242. chemistry.gsu.edu/glactone/PDB/ Proteins/Krebs/Krebs.html
44	Glyoxalate Pathway
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46	The Glyoxylate Cycle As we've seen fats are only used in biosynthesis for lipids in animals. Plants, on the other hand can use fats for biosynthesis of carbohydrates, amino acids, etc. Of course plants don't generally store lots of energy as fat, except in their mobile forms, such as seeds. Seeds then use this fat, which is a dense form of energy storage, to manufacture the carbohydrate and protein needed to sprout. So how do seeds use fat for biosynthesis? Plants adds two new enzyme activities to the set seen in the TCA Cycle to create a new pathway, the Glyoxylate Cycle or Pathway. The stoichiometry of this pathway is:
47	"2 Acetyl-SCoA + NAD⁺" 2 Acetyl-SCoA + NAD⁺ + FAD Æ Malate + NADH + H⁺ + FADH₂ The pathway can be represented by a simple cycle with two acetyl-SCoA's added with succinate as the product, the Glyoxylate Cycle. In actuality, the pathway is broken up into two parts by being compartmentalized in the mitochondria and a specialized organelle, the Glyoxysome. The two new reactions occur in this organelle:
48	Isocitrate lyase catalyzes the cleavage of isocitrate to give succinate and glyoxalate:
49	Malate synthase uses an Aldol condensation followed by hydrolysis of the SCoA thiolester bond to synthesize Malate (the Citrate synthase reaction, but substituting glyoxylate for oxalacetate):
50	What is Gluconeogenesis? Gluconeogenesis is the formation of glucose from non-carbohydrate precursors, such as pyruvate, lactate, certain amino acids, and intermediates of TCA cycle. Glyoxylate cycle is special example of gluconeogenesis that is specific to plants. It represents a shortcut, or shunt, across the TCA cycle. The following reactions summarize the chemistry of the glyoxylate cycle: a. isocitrate ---> succinate + glyoxylate b. glyoxylate + Ac-CoA ---> malate --> oxaloacetate c. oxaloacetate is exported from glyoxysome ---> to mitochondrion ---> gluconeogenesis
51	Gluconeogenesis is active when: a. high lactate levels from muscle activity (a product of anaerobic metabolism) b. starvation (starvation, in the biochemical sense, is due to lack off glucose not of food or ATP)
52	"Gluconeogenesis takes place in the..." Gluconeogenesis takes place in the cytosol of liver and the cortex of kidney (to lesser extent). It is the reversal of the reactions of the glycolytic pathway except for the three reactions that are highly exergonic, and hence not easily reversible. A set of alternate reactions circumvent these energy barriers, but they require the use of metabolic energy in order to proceed in the desired direction. One of these reactions is the conversion of pyruvate to phosphoenolpyruvate; in glycolysis the reaction is phosphoenolpyruvate + ADP --> pyruvate + ATP (catalyzed by pyruvate kinase) The reversal of this glycolytic reaction requires two reactions specific to gluconeogenesis:
53	a. Pyruvate carboxylase (an anaplerotic reaction that we have seen before) pyruvate + HCO3- + ATP ----> oxaloacetate + ADP + Pi Pyruvate carboxylase is completely inactive in absence of acetyl-CoA, which acts as a positive allosteric modulator. This type of regulation makes sense because high levels of Ac-CoA signal the need for more oxaloacetate. This oxaloacetate is formed inside the mitochondrion, and passes into the cytoplasm as malate: NADH + OAAm ----> malatem ----> malatec ----> OAAc For this reaction to occur, the mitochondrial levels of NADH must be high (this would occur if energy levels were also high). Acetate, itself, is not a precursor to glucose in animals, because they have no glyoxylate cycle.
54	b. Phosphoenolpyruvate carboxykinase A second reaction completes the conversion of pyruvate (now oxaloacetate) to phosphoenolpyruvate: oxaloacetate + GTP ----> phosphoenolpyruvate + GDP + CO2 The sum of the two reactions is pyruvate + ATP + GTP <===> PEP + ADP + GDP + Pi The DG' is - 25 kJ/mol (under cellular conditions), and will only proceed when ATP/ADP is high (this means that the cell can afford to make glucose).
55	c. Fructose bisphosphatase F 6-P + ATP ===> F 1,6-DP + ADP This glycolytic reaction is catalyzed by phosphofructokinase, which is activated by AMP, inhibited by citrate. In gluconeogenesis, the reverse reaction is catalyzed by fructose bisphosphatase, which is a cytosolic enzyme. F 1,6-DP + H2O <===> F 6-P + Pi DGo' = - 16.3 kJ/mol
56	"This enzyme is inhibited by..." This enzyme is inhibited by AMP (i.e. it requires a high energy state to be active), and it is stimulated by 3-phosphoglycerate and citrate (TCA cycle is proceeding slowly because there is no need for new ATP). Notice that the regulation of glycolysis and gluconeogenesis is complimentary. <> The liver expresses the gene for this enzyme, but muscle does not. Hence the liver can release glucose and the muscle can not. G-6-P ----> releases free glucose (goes to bloodstream and then to the brain)
57	Comparison of overall reactions Gluconeogenesis: 2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H2O ----> glucose + 4 ADP + 2 GDP + 2 NAD+ + 6 Pi DG = - 37.6 kJ/mole Glycolysis: glucose + 2 ADP + 2 Pi + 2 NAD+ ----> 2 pyruvate + 2 ATP + 2 NADH + 2 H2O DG = -83.7 kJ/mole = glycolysis or +83.7 kJ/mole if gluconeogenesis were the reverse of glycolysis (clearly, this can't happen) There is a loss of 4 moles of ATP/mole of glucose made by gluconeogenesis.
58	Review of Energy Physiology a. the brain requires glucose (it can use ketone bodies during starvation) b. muscles, when at rest, use fatty acids; when exercising they use glycogen and can produce lactate when oxygen levels are limiting. c. liver (the glucose buffer) converts lactate to glucose d. adipose tissue needs glucose for triglyceride synthesis; low glucose leads to release of fatty acids.
59	Ethanol and gluconeogenesis: Extreme alcoholics (winos) have very clean arteries (low risk of heart disease, stroke) but their livers are like stone (due to scarring). They are also very gaunt, due to loss of muscle mass (glucogenic amino acids are being converted to glucose, since ethanol can't participate in gluconeogenesis). Ethanol is metabolized in the human body via the enzyme alcohol dehydrogenase; the reaction sequence is as follows: ethanol ----> acetaldehyde ----> acetate Acetaldehyde is similar to formaldehyde, which is used as pickling agent. It builds up in this metabolic sequence because the second reaction is slower than the first (i.e. it is rate-limiting).
60	Regulation of gluconeogenesis: cAMP ---> stimulates the production of F-2,6- BP ---> slows gluconeogenesis glucagon ---> breakdown of glycogen ---> release of glucose