Bioenergetics The tiny hummingbirds can store enough fuel

Bioenergetics The tiny hummingbirds can store enough fuel to fly a distance of 500 miles without resting. This achievement is possible because of the ability to convert fuels into the cellular energy currency, ATP.

• Metabolism - the entire network of chemical reactions carried out by living cells. Metabolism also includes coordination, regulation and energy requirement. • Metabolites - small molecule intermediates in the degradation and synthesis of polymers Most organism use the same general pathway for extraction and utilization of energy. All living organisms are divided into two major classes: Autotrophs – can use atmospheric carbon dioxide as a sole source of carbon for the synthesis of macromolecules. Autotrophs use the sun energy for biosynthetic purposes. Heterotrophs – obtain energy by ingesting complex carboncontaining compounds. Heterotrophs are divided into aerobs and anaerobs.

Common features of organisms 1. Organisms or cells maintain specific internal concentrations of inorganic ions, metabolites and enzymes 2. Organisms extract energy from external sources to drive energy-consuming reactions 3. Organisms grow and reproduce according to instructions encoded in the genetic material 4. Organisms respond to environmental influences 5. Cells are not static, and cell components are continually synthesized and degraded (i. e. undergo turnover)

A sequence of reactions that has a specific purpose (for instance: degradation of glucose, synthesis of fatty acids) is called metabolic pathway. Metabolic pathway may be: (a) Linear (b) Cyclic (c) Spiral pathway (fatty acid biosynthesis)

Metabolic pathways can be grouped into two paths – catabolism and anabolism Catabolic reactions - degrade molecules to create smaller molecules and energy Anabolic reactions - synthesize molecules for cell maintenance, growth and reproduction Catabolism is characterized by oxidation reactions and by release of free energy which is transformed to ATP. Anabolism is characterized by reduction reactions and by utilization of energy accumulated in ATP molecules. Catabolism and anabolism are tightly linked together by their coordinated energy requirements: catabolic processes release the energy from food and collect it in the ATP; anabolic processes use the free energy stored in ATP to perform work.

Anabolism and catabolism are coupled by energy

Metabolism Proceeds by Discrete Steps • Multiple-step pathways permit control of energy input and output Single-step vs multi-step pathways • Catabolic multi-step pathways provide energy in smaller stepwise amounts • Each enzyme in a multistep pathway usually catalyzes only one single step in the pathway • Control points occur in multistep pathways A multistep enzyme pathway releases energy in smaller amounts that can be used by the cell

Metabolic Pathways Are Regulated • Metabolism is highly regulated to permit organisms to respond to changing conditions • Most pathways are irreversible • Flux - flow of material through a metabolic pathway which depends upon: (1) Supply of substrates (2) Removal of products (3) Pathway enzyme activities

Levels of Metabolism Regulation 1. Nervous system. 2. Endocrine system. 3. Interaction between organs. 4. Cell (membrane) level. 5. Molecular level

Feedback inhibition • Product of a pathway controls the rate of its own synthesis by inhibiting an early step (usually the first “committed” step (unique to the pathway) Feed-forward activation • Metabolite early in the pathway activates an enzyme further down the pathway

Covalent modification for enzyme regulation • Interconvertible enzyme activity can be rapidly and reversibly altered by covalent modification • Protein kinases phosphorylate enzymes (+ ATP) • Protein phosphatases remove phosphoryl groups

Regulatory role of a protein kinase, amplification by a signaling cascade The initial signal may be amplified by the “cascade” nature of this signaling

Stages of metabolism Catabolism Stage I. Breakdown of macromolecules (proteins, carbohydrates and lipids to respective building blocks. Stage II. Amino acids, fatty acids and glucose are oxidized to common metabolite (acetyl Co. A) Stage III. Acetyl Co. A is oxidized in citric acid cycle to CO 2 and water. As result reduced cofactor, NADH 2 and FADH 2, are formed which give up their electrons. Electrons are transported via the tissue respiration chain and released energy is coupled directly to ATP synthesis.

Glycerol Catabolism

Catabolism is characterized by convergence of three major routs toward a final common pathway. Different proteins, fats and carbohydrates enter the same pathway – tricarboxylic acid cycle. Anabolism can also be divided into stages, however the anabolic pathways are characterized by divergence. Monosaccharide synthesis begin with CO 2, oxaloacetate, pyruvate or lactate. Amino acids are synthesized from acetyl Co. A, pyruvate or keto acids of Krebs cycle. Fatty acids are constructed from acetyl Co. A. On the next stage monosaccharides, amino acids and fatty acids are used for the synthesis of polysaccharides, proteins and fats.

Compartmentation of Metabolic Processes in Cell • Compartmentation of metabolic processes permits: - separate pools of metabolites within a cell - simultaneous operation of opposing metabolic paths - high local concentrations of metabolites • Example: fatty acid synthesis enzymes (cytosol), fatty acid breakdown enzymes (mitochondria)

Compartmentation of metabolic processes

The chemistry of metabolism There about 3000 reactions in human cell. All these reactions are divided into six categories: 1. Oxidation-reduction reactions 2. Group transfer reactions 3. Hydrolysis reactions 4. Nonhydrolytic cleavage reactions 5. Isomerization and rearrangement reactions 6. Bond formation reactions using energy from ATP

1. Oxidation-reduction reactions are those in which electrons are transferred from one molecule or atom to another Enzymes: oxidoreductases -oxidases - peroxidases - dehydrogenases -oxigenases Coenzymes: NAD+, NADP+, FAD+, FMN+ Example:

2. Group transfer reactions Transfer of a chemical functional group from one molecule to another (intermolecular) or group transfer within a single molecule (intramolecular) Enzymes: transferases Examples: Phosphorylation Acylation Glycosylation

3. Hydrolysis reactions • Water is used to split the single molecule into two molecules Enzymes: hydrolases - esterases - peptidases - glycosidases Example:

4. Nonhydrolytic cleavage reactions • Split or lysis of a substrate, generating a double bond in a nonhydrolytic (without water), nonoxidative elimination Enzymes: lyases Example:

5. Isomerization and rearrangement reactions Two kinds of chemical transformation: 1. Intramolecular hydrogen atom shifts changing the location of a double bond. 2. Intramolecular rearrangment of functional groups. Enzymes: isomerases Example:

6. Bond formation reactions using energy from ATP • Ligation, or joining of two substrates • Require chemical energy (e. g. ATP) Enzymes: ligases (synthetases)

Experimental Methods for Studying Metabolism • Add labeled substrate to tissues, cells, and follow emergence of intermediates. Use sensitive isotopic tracers (3 H, 14 C etc) • Verify pathway steps in vitro by using isolated enzymes and substrates • Study of the mutations in genes associated with the production of defective enzymes • Use metabolic inhibitors to identify individual steps and sequence of enzymes in a pathway

OXIDATIVE DECARBOXYLATION OF PYRUVATE Matrix of the mitochondria contains pyruvate dehydrogenase complex

The fate of glucose molecule in the cell Synthesis of glycogen Glucose-6 phosphate Glycogen Pentose phosphate pathway Ribose, NADPH Degradation of glycogen Gluconeogenesis Glycolysis Ethanol Pyruvate Acetyl Co A Lactate

OXIDATIVE DECARBOXYLATION OF PYRUVATE Only about 7 % of the total potential energy present in glucose is released in glycolysis. Glycolysis is preliminary phase, preparing glucose for entry into aerobic metabolism. Pyruvate formed in the aerobic conditions undergoes conversion to acetyl Co. A by pyruvate dehydrogenase complex. Pyruvate dehydrogenase complex is a bridge between glycolysis and aerobic metabolism – citric acid cycle. Pyruvate dehydrogenase complex and enzymes of cytric acid cycle are located in the matrix of mitochondria.

Entry of Pyruvate into the Mitochondrion Pyruvate freely diffuses through the outer membrane of mitochondria through the channels formed by transmembrane proteins porins. Pyruvate translocase, protein embedded into the inner membrane, transports pyruvate from the intermembrane space into the matrix in symport with H+ and exchange (antiport) for OH-.

Conversion of Pyruvate to Acetyl Co. A • Pyruvate dehydrogenase complex (PDH complex) is a multienzyme complex containing 3 enzymes, 5 coenzymes and other proteins. Pyruvate dehydrogenase complex is giant, with molecular mass ranging from 4 to 10 million daltons. Electron micrograph of the pyruvate dehydrogenase complex from E. coli.

Enzymes: E 1 = pyruvate dehydrogenase E 2 = dihydrolipoyl acetyltransferase E 3 = dihydrolipoyl dehydrogenase Coenzymes: TPP (thiamine pyrophosphate), lipoamide, HS-Co. A, FAD+, NAD+. TPP is a prosthetic group of E 1; lipoamide is a prosthetic group of E 2; and FAD is a prosthetic group of E 3. The building block of TPP is vitamin B 1 (thiamin); NAD – vitamin B 5 (nicotinamide); FAD – vitamin B 2 (riboflavin), HS-Co. A – vitamin B 3 (pantothenic acid), lipoamide – lipoic acid

Pyruvate dehydrogenase complex is a classic example of multienzyme complex Overall reaction of pyruvate dehydrogenase complex The oxidative decarboxylation of pyruvate catalized by pyruvate dehydrogenase complex occurs in five steps.

Aerobic cells use a metabolic wheel – the citric acid cycle – to generate energy by acetyl Co. A oxidation The Citric Acid Cycle

Synthesis of glycogen Glucose Pentose phosphate pathway Glucose-6 phosphate Glycogen Ribose, NADPH Degradation of glycogen Gluconeogenesis Glycolysis Ethanol Fatty Acids The citric acid cycle is the final common pathway for the oxidation of fuel molecules — amino acids, fatty acids, and carbohydrates. Pyruvate Lactate Acetyl Co A Amino Acids Most fuel molecules enter the cycle as acetyl coenzyme A.

Names: The Citric Acid Cycle Tricarboxylic Acid Cycle Krebs Cycle In eukaryotes the reactions of the citric acid cycle take place inside mitochondria Hans Adolf Krebs. Biochemist; born in Germany. Worked in Britain. His discovery in 1937 of the ‘Krebs cycle’ of chemical reactions was critical to the understanding of cell metabolism and earned him the 1953 Nobel Prize for Physiology or Medicine.

An Overview of the Citric Acid Cycle A four-carbon oxaloacetate condenses with a two-carbon acetyl unit to yield a six-carbon citrate. An isomer of citrate is oxidatively decarboxylated and five-carbon ketoglutarate is formed. -ketoglutarate is oxidatively decarboxylated to yield a four-carbon succinate. Oxaloacetate is then regenerated from succinate. Two carbon atoms (acetyl Co. A) enter the cycle and two carbon atoms leave the cycle in the form of two molecules of carbon dioxide. Three hydride ions (six electrons) are transferred to three molecules of NAD+, one The function of the citric acid pair of hydrogen atoms (two electrons) is cycle is the harvesting of highenergy electrons from acetyl Co. A. transferred to one molecule of FAD.

1. Citrate Synthase • Citrate formed from acetyl Co. A and oxaloacetate • Only cycle reaction with C-C bond formation • Addition of C 2 unit (acetyl) to the keto double bond of C 4 acid, oxaloacetate, to produce C 6 compound, citrate synthase

2. Aconitase • Elimination of H 2 O from citrate to form C=C bond of cis-aconitate • Stereospecific addition of H 2 O to cis-aconitate to form isocitrate aconitase

3. Isocitrate Dehydrogenase • Oxidative decarboxylation of isocitrate to a-ketoglutarate (a metabolically irreversible reaction) • One of four oxidation-reduction reactions of the cycle • Hydride ion from the C-2 of isocitrate is transferred to NAD+ to form NADH • Oxalosuccinate is decarboxylated to a-ketoglutarate isocitrate dehydrogenase

4. The -Ketoglutarate Dehydrogenase Complex • Similar to pyruvate dehydrogenase complex • Same coenzymes, identical mechanisms E 1 - a-ketoglutarate dehydrogenase (with TPP) E 2 – dihydrolipoyl succinyltransferase (with flexible lipoamide prosthetic group) E 3 - dihydrolipoyl dehydrogenase (with FAD) a-ketoglutarate dehydrogenase

5. Succinyl-Co. A Synthetase • Free energy in thioester bond of succinyl Co. A is conserved as GTP or ATP in higher animals (or ATP in plants, some bacteria) • Substrate level phosphorylation reaction + Succinyl-Co. A Synthetase GTP + ADP GDP + ATP HS-

6. The Succinate Dehydrogenase Complex • Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters • Embedded in the inner mitochondrial membrane • Electrons are transferred from succinate to FAD and then to ubiquinone (Q) in electron transport chain • Dehydrogenation is stereospecific; only the trans isomer is formed Succinate Dehydrogenase

7. Fumarase • Stereospecific trans addition of water to the double bond of fumarate to form L-malate • Only the L isomer of malate is formed Fumarase

8. Malate Dehydrogenase Malate is oxidized to form oxaloacetate. Malate Dehydrogenase

Stoichiometry of the Citric Acid Cycle § Two carbon atoms enter the cycle in the form of acetyl Co. A. § Two carbon atoms leave the cycle in the form of CO 2. § Four pairs of hydrogen atoms leave the cycle in four oxidation reactions (three molecules of NAD+ one molecule of FAD are reduced). § One molecule of GTP, is formed. § Two molecules of water are consumed. § 9 ATP (2. 5 ATP per NADH, and 1. 5 ATP per FADH 2) are produced during oxidative phosphorylation. § 1 ATP is directly formed in the citric acid cycle. § 1 acetyl Co. A generates approximately 10 molecules of ATP.

Functions of the Citric Acid Cycle • Integration of metabolism. The citric acid cycle is amphibolic (both catabolic and anabolic). The cycle is involved in the aerobic catabolism of carbohydrates, lipids and amino acids. Intermediates of the cycle are starting points for many anabolic reactions. • Yields energy in the form of GTP (ATP). • Yields reducing power in the form of NADH 2 and FADH 2.

Regulation of the Citric Acid Cycle • Pathway controlled by: (1) Allosteric modulators (2) Covalent modification of cycle enzymes (3) Supply of acetyl Co. A (pyruvate dehydrogenase complex) Three enzymes have regulatory properties - citrate synthase (is allosterically inhibited by NADH, ATP, succinyl Co. A, citrate – feedback inhibition) - isocitrate dehydrogenase (allosteric effectors: (+) ADP; (-) NADH, ATP. Bacterial ICDH can be covalently modified by kinase/phosphatase) - -ketoglutarate dehydrogenase complex (inhibition by ATP, succinyl Co. A and NADH

Regulation of the citric acid cycle - NADH, ATP, succinyl Co. A, citrate

Krebs Cycle is a Source of Biosynthetic Precursors Glucose Phosphoenolpyruvate The citric acid cycle provides intermediates for biosyntheses