CITRIC ACID (KREB S, TCA) CYCLE

Transcription

1 ITRI AID (KREB S, TA) YLE Date: September 2, 2005 * Time: 10:40 am 11:30 am * Room: G202 Biomolecular Building Lecturer: Steve haney 515A Mary Ellen Jones Building stephen_chaney@med.unc.edu *Please consult the online schedule for this course for the definitive date and time for this lecture. ffice Hours: by appointment Assigned Reading: This syllabus. Key oncepts vs Supplementary Information: Because this syllabus is meant to replace the need for a Biochemistry textbook, it contains a mixture of information that is critical for you to know and information that serves to illustrate and explain the key points. I have attempted to emphasize important terms, definitions and concepts in red, and have listed key points after each section of the syllabus. Illustrative and supplementary information is indicated in italics. verall bjectives for arbohydrate Metabolism: By the end of this course, you should: 1) understand how carbohydrate metabolism normally responds in the fed state, the fasting state, and during exercise. 2) understand how carbohydrate metabolism is altered by diabetes and the metabolic response to trauma and surgery. 3) understand the relationship between obesity, exercise, insulin resistance, and diabetes. 4) understand basic priciples of diet composition and weight management. Lecture bjectives: At the conclusion of this lecture you should: 1) know the vitamins and cofactors that are required by the citric acid cycle (especially for the pyruvate dehydrogenase and αketoglutarate dehydrogenase reactions). 2) know how the citric acid cycle is regulated. 3) know the role that the citric acid cycle plays in carbohydrate, amino acid, and fat metabolism. 4) know the role that the citric acid cycle plays in the interconversion of carbohydrates, amino acids, and fats.

2 Important concepts: 1. All fuel molecules are oxidized to citric acid cycle intermediates. 2. The citric acid cycle is important for the biosynthesis of glucose, lipids, and some amino acids. The citric acid cycle breaks down a 6 carbon compound (citrate) to a 4 carbon compound (oxaloacetate). The high energy electrons are transferred to coenzyme carriers (NAD and FAD) and destined for oxidative phosphorylation. The carbons are removed as, and the H will follow with the electrons to 2 to form H 2. The citric acid cycle enzymes are located in the mitochondria, but they are mostly soluble enzymes located in the mitochondrial matrix. A. Pyruvate dehydrogenase complex (PDH) Pyruvate dehydrogenase is a large enzyme complex in the mitochondrion consisting of 3 different types of enzyme subunits. It is the enzyme that connects the glycolytic pathway to the citric acid cycle. onnects Glycolysis to.a.. oash NADH SoA H 3 NAD H 3 pyruvate acetyl oa Note that 4 of the coenzymes used in this reaction are derived from substances that must be obtained in the diet. thiamine or vitamin B 1 thiamine pyrophosphate (TPP) pantothenic acid coenzyme A (oash) riboflavin FADH 2 niacin NADH

3 B. verview of the itric Acid ycle pyruvate H3 oash pyruvate NADH dehydrogenase SoA 2 H3 acetyl oa oash H 2 H NADH itrate synthase 2 AA citric HH Malate acid dehydrogenase NADH Succinyl oa synthetase malate H 2 H NADH αkg 2 oash SoA 2 oash αkg dehydrogenase succinate GTP succinyl oa FADH 2 The citric acid cycle contains a cyclic sequence of 8 enzymes that are so arranged that they perform molecular rearrangements of intermediate metabolic compounds to prepare them for decarboxylation and dehydrogenation..selected Enzymatic Steps 1. itrate synthase: this is a 2 step reaction: an aldol condensation of oxaloacetate and acetyl oa, followed by hydrolysis to yield citrate and free oa. The hydrolysis step is not easily reversible. SoA H3 acetyl oa itrate synthase oash H citric acid

4 2. αketoglutarate dehydrogenase: a multienzyme complex very similar to pyruvate dehydrogenase. This step is irreversible. It also produces NADH. NADH αkg SoA succinyl oa αkg dehydrogenase oash 3. succinyl oa synthetase: succinyl oa has a high negative ΔG ' of hydrolysis, and can, therefore, be coupled to the direct phosphorylation of GDP GTP (which is equivalent to ATP); this reaction is fairly reversible. This is an example of substrate level phosphorylation. succinate Succinyl oa synthetase oash GTP SoA succinyl oa 4. malate dehydrogenase: this is a good example of a reaction that has a net flow opposite to an unfavorable Keq. That is the oxidation of malate by NAD to produce oxaloacetate NADH H has a G ' of 7 kcal/mole. NADH HH AA Malate dehydrogenase malate Question: What can you deduce from the above direction of this reaction about the respective physiological concentrations of malate and oxaloacetate?

5 Net Energy Yield of the itric Acid ycle For each acetyl group used up, the cycle produces: 3 NADH 1 FADH 2 1 GTP 2 Since each NADH can result in 3 ATP, and each FADH 2 can result in 2 ATP, the net high energy production is 12 ATP. Therefore, the total energy from 1 glucose is: 1 glucose 2 pyruvate: 2 ATP 2 NADH (= 4 ATP) 2 pyruvate 2 acetyl oa: 2 NADH 2 acetyl oa 4 : 2 x 12 ATP 6 ATP 6 ATP 24 ATP 6 in cytosol 30 in mitochondria 36 ATP It should be selfevident why the 36 ATP that result from the complete oxidation of glucose are far preferable to the 2 ATP that are derived by glycolysis alone.. Regulation of the itric Acid ycle The citric acid cycle is regulated at multiple points. However, in general it is safe to say that it is inhibited by ATP and NADH. The inhibition by NADH keeps it tightly regulated by oxygen supply, since NADH is converted to NAD by oxidative phosphorylation. The inhibition by ATP keeps the citric acid cycle in balance with energy supply. When ATP (energy supply) is high, the citric acid cycle is inhibited and precursors to the citric acid cycle (pyruvate, acetyl oa and amino acids) are diverted into other pathways. Acetyl oa, citrate, and succinyloa are the end products of individual steps in the citric acid cycle and their accumulation inhibits the step involved in their production. That, of course, results in inhibition of the cycle as a whole. Finally, a stimulates the citric acid cycle at several points. This is important because electrical stimulation of the muscle causes an increase in intracellular calcium levels. Thus, during exercise the citric acid cycle will be maximally stimulated in muscle. The regulation of the citric acid cycle is summarized below.

6 Pyruvate a 2 e AcetyloA xaloacetate Malate Fumarate Succinate GTP SuccinyloA ATP NADH itrate Isocitrate αketoglutarate e a 2 a 2 e x Phos Regulation of the itric Acid ycle (animation available in PowerPoint) D. Relationship Between the itric Acid ycle and ther Metabolic Pathways 1. Acetyl oa is both the final product of fatty acid degradation and the first building block for fatty acid synthesis. 2. Several citric acid cycle intermediates can be converted to amino acids by simple transamination reactions (see below). Thus, the citric acid cycle can be involved in amino acid synthesis, degradation, or conversion to oxaloacetate for gluconeogenesis.

7 αkg Asp Glu AA H NH 3 H NH 3 αketoglutarate Aspartate Glutamate xaloacetate.a.. and ther Pathways Glucose protein a.a. s PEP Alanine protein pyruvate fats a.a. s fatty 2 acetyl oa acids Asp citrate ( 6 ) AA ( 4 ) αkg ( 5 ) protein a.a. s NADH FADH 2 ATP succinate ( 4 ) Val, Ile porphyrins protein hemoglobin glutamate urea cycle Interconversions of the itric Acid ycle (animation available in PowerPoint)

8 Thus, the citric acid cycle: 1. is the final step in the conversion of all foods to and H is the major source of reducing equivalents (NADH and FADH 2 ) used by the cell to generate ATP (via oxidative phosphorylation). 3. is the central pathway that interconnects all others. a. Excess carbohydrate can be converted to protein* and fat. b. Excess protein can be converted to carbohydrate or fat. c. However, net conversion of fatty acids to carbohydrate and most amino acids is not possible. *Note: Some amino acids cannot be synthesized by the body and are, therefore, essential components of the diet. In the absence of these essential amino acids, net conversion of carbohydrate to complete proteins is impossible. 4. The citric acid cycle requires a constant supply of oxaloacetate to keep going. (What enzyme supplies oxaloacetate when we break down carbohydrate?) (Why is this enzyme activated by acetyl oa?) 5. Since oxaloacetate is depleted by gluconeogenesis, Dr. Atkins has postulated that fat calories won t be utilized in the absence of carbohydrate. Based on what you now know about the citric acid cycle, what is the basic falacy of this hypothesis? (Where does oxaloacetate come from when we break down protein?)

9 Why Does Atkins Diet Appear to Work? H H 2 retention (short term) long term: wt loss = caloric balance high H weight high fat time Any healthy diet will do Main argument against Atkins = unhealthy diet Low carbohydrate versus low fat diets will be covered in more detail in lecture. Key Points about the itric Acid ycle 1. ofactors for the pyruvate dehydrogenase reaction and their corresponding vitamins. thiamine pyrophosphate lipoic acid coenzyme A FADH 2 NAD thiamin pantothenic acid riboflavin niacin 2. Regulation of.a.. When 2 limiting it causes an increase in [NADH] which causes inhibition (NADH = direct end product.a..).

10 When cell has more energy than it needs the concentration of [ATP] increases, which causes inhibition. ATP = end product.a.. and ox. phos. This diverts pyruvate, acetyl oa and amino acids to other pathways 3..A.. and ther Pathways It is the final step for conversion of all foods to and H 2. It is the central pathway that interconnects all others. H can be converted to protein or fat. Some protein can be converted to H or fat. Fat cannot be converted to H or protein. AA is needed to keep the.a.. going. SAMPLE QUESTINS Each of the following questions has one correct answer. 1. The overall reaction of the pyruvate dehydrogenase complex produces: NADH, H,, and a. lactate b. oxaloacetate c. citrate d. ATP e. Acetyl oa 2. The citric acid cycle and oxidative phosphorylation both occur in the: a. lysosomes b. nucleus c. zymogen granules d. cytoplasm e. mitochondrion

11 3. The citric acid cycle "begins" with citrate synthase catalyzing the formation of citrate from: a. succinyl oa pyruvate b. acetyl oa pyruvate c. acetyl oa oxaloacetate d. furmarate oxaloacetate e. pyruvate lactate

12 Answers 1. e 2. e 3. c omments 1. Pyruvate dehydrogenase is a key enzyme. You are, therefore, asked to remember that it converts pyruvate to acetyloa. 2. You need to know the location of metabolic pathways. e is correct. 3. Again this is a key enzyme. You are asked to remember the substrates.