INTRODUCTION TO METABOLISM What is Metabolism?

This is the sum total of the biochemical reactions which take place within a cell. The biochemical reactions occur through a series of enzymecatalyzed reactions known as metabolic pathways. Each of the consecutive steps in a metabolic pathway brings about a specific, small chemical change, usually the removal, transfer, or addition of a particular atom or functional group. Metabolic processes do not include single functions (or processes) such as protein-protein interactions, protein-nucleic acids, recepter-ligand interactions, etc The term intermediary metabolism is often applied to the combined activities of all the metabolic pathways that interconvert precursors, metabolites, and products of low molecular weight. Functions of Metabolism i. Breakdown complex molecules (nutrients) into precursors molecules. ii. Capture the energy released from nutrient breakdown. iii. Synthesis of necessary molecules Metabolism can classified as: i. Catabolism ii. Anabolism Catabolism is the degradative phase of metabolism in which organic nutrient molecules (carbohydrates, fats, and proteins) are converted into smaller, simpler end products (such as lactic acid, CO2 , NH3). as heat. Catabolic pathways release energy, some of which is conserved in the

formation of ATP and reduced electron carriers (NADH, NADPH, and FADH2); the rest is lost

In anabolism, also called biosynthesis, small, simple precursors are built up into larger and more complex molecules, including lipids, polysaccharides, proteins, and nucleic acids. Anabolic reactions require an input of energy, generally in the form of the phosphoryl group transfer potential of ATP, precursor molecules and the reducing power of NADH, NADPH, and FADH2(All from catabolic reactions).

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Metabolic pathways These are hundreds of highly specific individual reactions taking place within a cell. These reactions proceed at very high rates in physiological conditions. Most of the chemical interconversions taking place within a cell involve sequences of linked reactions.

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Linked sequences of biochemical reactions make up metabolic pathways, i.e., the product of one reaction is the substrate for the next. The individual molecules which are converted from one molecule to another, through these pathways, are known as Metabolites.

The molecules between the initial substrate and the final product are called metabolic intermediates. For example, glucose-6-phosphate and glyceraldehyde 3-phosphate are examples of metabolic intermediates of glycolysis.

Metabolic pathways can be linear (glycolysis) or cyclic (TCA cycle) sequences of reactions. Metabolic pathways are often branched i.e., intermediates can be siphoned off and used as substrates for other pathways. The product of one pathway can be the starting substrate for another pathway.

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Energy exchange between pathways The oxidation of carbohydrates, fats and proteins during catabolism liberates substantial amounts of free energy. The direct interaction of these molecules with molecular oxygen would release this energy as heat (which is not useful). These molecules are oxidised in many small steps (useful release of energy).

The energy liberated in some of these smaller steps is used to synthesise ATP from ADP and P i. This captures a considerable portion of the free energy released by these metabolic conversions. This energy can be used for energy requiring processes.

ATP is the link molecule/coupling agent between catabolic and anabolic reactions. The ratio of ADP/ATP within a cell is kept reasonably constant, i.e., ATP is not stored and almost as soon as it is synthesized it is used up. Cells store energy in the form of energy-rich nutrients (triacylglycerols and glycogen). Energy-rich nutrients Excess carbohydrate is stored in the liver in the form of glycogen. Fats are stored as

triacylglycerol in adipose tissue. Amino acids are not stored and excess amino acids are broken down to yield precursor molecules and the amino groups are excreted in the form of urea. Regulation of metabolism Cells must tightly regulate the different metabolic pathways. For example, If a cell is short of energy is oxidises glucose the generate energy. It would be pointless converting the glucose to glycogen. The flow of metabolites through a metabolic pathway depends on: Med. Biochemistry notes by Musyoki [MPCU] Page 4

i. the concentration of the substrates ii. the amount of product formed iii. the activity of the enzymes catalysing the different steps. The level of substrates is usually kept fairly constant. As soon as a product is formed it is rapidly used up by another enzyme (as in a metabolic pathway) or removed from the site of the reaction. The most effective way to regulate metabolic pathways is to regulate the activity of the enzymes of that pathway. Regulation of enzyme activity This is accomplished in several mechanisms i. Covalent modifications Phosphorylation of enzymes can lead to activation or inhibition depending on the enzyme. ii. Allosteric control Enzymes may have a binding site for an activator or an inhibitor which alters the activity of the enzyme. iii. Regulation of enzyme synthesis It is Slow and requires energy. iv. Compartmentation This is whereby enzymes and substrates are held in a confined space. v. Feedback inhibition vi. Feed forward activation Most of the reactions in living cells fall into one of five general categories: i. Example Reduction-oxidation (Redox) reactions

ii.

Reactions that make or break carboncarbon bonds Example

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iii.

Internal rearrangements, isomerizations, and eliminations Example:

iv.

Group transfer reactions Example:

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v.

Free radical reactions Example:

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RESPIRATORY CHAIN AND OXIDATIVE PHOSPHORYLATION

The Metabolic Pathway of Cellular Respiration All of the reactions involved in cellular respiration can be grouped into three main stages i) Glycolysis occurs in cytoplasm ii) The Krebs cycle occurs in the matrix of mitochondria iii) Electron transport occurs across the mitochondrial membrane Mitochondrial Structure

The mitochondrion is the power house of the cell and generates the ATP used as energy by the cell. It is the site of the major energy producing cellular processes including TCA cycle, fatty acid oxidation and oxidative phosphorylation. 1. Cristae formed by the large folds in the inner membrane and increases the surface area for maximizing the amount of inner membrane bound metabolic enzymes that can exist in one mitochondrion. 2. Outer membrane is simple in protein composition and serves to maintain the shape of the mitochondrion. It is freely permeable to small molecules. 3. Matrix contains the enzymes of the TCA cycle with the exception of succicinate dehydrogenase, which is found on the inner membrane with its active site facing the matrix. The enzymes of fatty acid oxidation are located in the matrix. In addition, the matrix contains mitochondrial DNA, ribosomes and other proteins required for synthesis of proteins encoded by the mitochondrial genome. 4. Inner membrane is more complex than outer membrane and richly packed with large variety of proteins, quite impermeable to molecules and ions.It is freely permeable only to O2, CO2, and H2O. Molecules that need to cross the membrane do so by specific transport proteins. It is extremely folded to form the cristae. 5. Intermediate space located between the inner and outer membranes. Several enzymes that utilize ATP, such as creatine kinase, and adenylate kinase, are inside here. Because of the extreme permeability of the outer membrane to small molecules such as metabolites, for the purpose of discussing metabolism the intermembrane space is equivalent to the cytosol. What is Oxidative phosphorylation? It is the production of ATP by way of an electron transport chain and using oxygen as the final electron acceptor. Med. Biochemistry notes by Musyoki [MPCU] Page 8

What is respiratory chain? A mechanism by which electrons are passed along a series of carrier molecules, releasing energy for the synthesis of ATP. Respiratory chain is also known as electron chain transport. The electron chain transport and oxidative phosphorylation processes take place on the inner membrane of the mitochondrion. The Electron chain transport consists of 4 protein complexes (I, II, III, and IV), that move the electrons from reduced electron carriers such as NADH and FADH2 to complex V, where oxidative phosphorylation takes place, generating ATP. The electrons move through a chain of donors and acceptors. The electrons flow down a gradient i.e. electrons move from a carrier with low reduction potential (high tendency to donate electrons) toward carriers with higher reduction potential (high tendency to accept electrons). Eventually the redox reactions of the respiratory chain use electrons to reduce oxygen to water. The energy generated at various steps moves protons from matrix to intermembrane space. The inward movement of protons from the intermembrane space to the matrix recovers this energy with the formation of ATP in the matrix. Coenzymes and cytochromes in the complexes act as electron donors & acceptors

The electron transport chain showing respiratory complexes

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NADH-coenzyme Q oxidoreductase (complex I) NADH-coenzyme Q oxidoreductase, also known as NADH dehydrogenase or complex I, is the first protein in the electron transport chain. The reaction that is catalyzed by this enzyme is the two electron oxidation of NADH by coenzyme Q or ubiquinone The start of the reaction, and indeed of the entire electron chain, is the binding of a NADH molecule to complex I. NADH Dehydrogenase accepts 2e-- from NADH and transfers them to ubiquinone (coenzyme Q), an electron carrier. It uses two bound cofactors to accomplish this: FMN (Flavin mono nucleotide) and iron-sulfur (Fe-S) protein. As the electrons pass through this complex, four protons are pumped from the matrix into the intermembrane space

Succinate-Q oxidoreductase (complex II) Succinate-Q oxidoreductase, also known as complex II or Succinate dehydrogenase, is a second entry point to the electron transport chain. It is unusual because it is the only enzyme that is part of both the citric acid cycle and the electron transport chain. Complex II consists of a bound Flavin adenine dinucleotide (FAD) cofactor and ironsulfur clusters. It oxidizes Succinate to fumarate and reduces ubiquinone. As this reaction releases less energy than the oxidation of NADH, complex II does not transport protons across the membrane and does not contribute to the proton gradient.

Q-cytochrome c oxidoreductase (complex III) Q-cytochrome c oxidoreductase is also known as cytochrome c reductase, cytochrome bc1 complex, or simply complex III. The reaction catalyzed by complex III is the oxidation of one molecule of ubiquinol and the reduction of two molecules of cytochrome c. Unlike coenzyme Q, which carries two electrons, cytochrome c carries only one electron. As the electrons pass through this complex, four protons are pumped from the matrix into the intermembrane space

Cytochrome c oxidase (complex IV) Cytochrome c oxidase, also known as complex IV, is the final protein complex in the electron chain transport. This enzyme mediates the final reaction in the electron transport chain and transfers electrons to oxygen, while pumping protons across the membrane. The final electron acceptor oxygen, which is also called the terminal electron acceptor, is reduced to water in this step. Both the direct pumping of protons and the consumption of matrix protons in the reduction of oxygen contribute to the proton gradient. The reaction catalyzed is the oxidation of cytochrome c and the reduction of oxygen:

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ATP Synthase (complex V) ATP synthase, also called complex V, is the final enzyme in the oxidative phosphorylation pathway. This enzyme is found in all forms of life and functions in the same way in both prokaryotes and eukaryotes. The enzyme uses the energy stored in a proton gradient across a membrane to drive the synthesis of ATP from ADP and phosphate (Pi). Estimates of the number of protons required to synthesize one ATP have ranged from three to four. This phosphorylation reaction is an equilibrium, which can be shifted by altering the protonmotive force. In the absence of a proton-motive force, the ATP synthase reaction will run from right to left, hydrolyzing ATP and pumping protons out of the matrix across the membrane. However, when the proton-motive force is high, the reaction is forced to run in the opposite direction; it proceeds from left to right, allowing protons to flow down their concentration gradient and turning ADP into ATP NAD+, FMN, CQ are carriers of electrons and hydrogen while cytochromes are carriers of electrons only.

Remember
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations. Cofactors are either organic or inorganic. They can also be classified depending on how tightly they bind to an enzyme, with loosely-bound cofactors termed coenzymes and tightly-bound cofactors termed prosthetic groups. Some sources also limit the use of the term "cofactor" to inorganic substances. An inactive enzyme, without the cofactor is called an apoenzyme, while the complete enzyme with cofactor is the holoenzyme. Heme is a prosthetic group of cytochromes. Heme contains an iron atom embedded in

a porphyrin ring system. Cytochromes are proteins with heme prosthetic groups.
Electron carriers alternately become oxidized and reduced during electron and proton transfer. The electron carriers include three freely diffusible coenzymes known as NAD+, FAD and NADP+. The reduced forms of these coenzymes (NADH, FADH2 and NADPH have reducing powers. NAD+ or Nicotinamide adenine dinucleotide, is a coenzyme that often works in conjunction with an enzyme called a dehydrogenase. The enzyme removes two hydrogen atoms (2H+ and 2e-) from its substrate. Both electrons but only one proton are accepted by the NAD+ to produce its reduced form, NADH, plus H+. NADH is used to generate proton motive force that can drive the synthesis of ATP. FAD or Flavin adenine dinucleotide, is a coenzyme that also works in conjunction with an enzyme called a dehydrogenase. The enzyme removes two hydrogen atoms (2H+ and 2e-) from its substrate. Both electrons and both protons are accepted by the FAD to produce its reduced Med. Biochemistry notes by Musyoki [MPCU] Page 11

form, FADH2. FADH2 is used to generate proton motive force that can drive the synthesis of ATP. NADP+, or nicotinamide adenine dinucleotide phosphate, is a coenzyme that uses dehydrogenase to remove two hydrogen atoms (2H+ and 2e-) from its substrate. Both electrons but only one proton are accepted by the NADP+ to produce its reduced form, NADPH, plus H+. NADPH is not used for ATP synthesis but its electrons provide the energy for certain biosynthesis reactions such as ones involved in photosynthesis.

Inhibitors of ATP synthesis

There are several well-known drugs and poisons that inhibit oxidative phosphorylation. They include; Barbiturates such as amytal and the plant toxin retinone used as pesticide inhibit transfer of electrons to coenzyme Q from complex I. The antibiotic Antimycin A inhibits transfer from complex III to cytochrome c. Cyanide and H2S which are poisons inhibit cytochrome c oxidase. Carbon monoxide inhibits cytochrome oxidase but its main action is inhibition of hemoglobin oxygen binding.

2,4 Dininitrophenol a poison uncouples proton gradient from ATP synthesis, because it can carry protons across the mitochondrial inner membrane. It was used as a weightloss drug. Although it was highly effective, it was also potentially lethal and is no longer used for dieting Regulation of the Electron transport pathway The synthesis of ATP by electron transport and oxidative phosphorylation appears to be regulated essentially exclusively by substrate availability. The pathway cannot proceed without ADP + Pi or NADH; if both are available ,then the pathway will result in ATP synthesis. HIGH ENERGY COMPOUNDS

Certain compounds are encountered in the biological systems which, on hydrolysis, yields energy. The term high-energy compounds or energy rich compounds are usually applied to substances which possess sufficient free energy to liberate at least 7 Cal/mol at pH 7.0. Certain other compounds which liberate less than 7 Cal/mol (lower than ATP hydrolysis to ADP + Pi) (table 1) are referred to as low- energy compounds.

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Table 1 Standard free energy of hydrolysis of some important compounds

Compounds Go (Cal/mol) High Energy Phosphates Phosphoenol pyruvate - 14.8 Carbamoyl phosphate - 12.3 Cyclic AMP - 12.0 1,3 Bisphosphoglycerate - 11.8 Phosphocreatine - 10.3 Acetyl phosphate - 10.3 S Adenosylmethionine - 10.0 Pyrophosphate - 8.0 Acetyl CoA - 7.7 - 7.3 ATPADP + Pi Low energy compounds ADPAMP + Pi - 6.6 Glucose 1-Phosphate - 5.0 Fructose 6-Phosphate - 3.8 Glucose 6-Phosphate - 3.3 Glycerol 3-Phosphate - 2.2 All the high energy compounds when hydrolyzed liberate more energy than that of ATP.Most of high energy compounds contain phosphate group (exception acetyl CoA) hence they are also called high energy phosphates.

Classification of high energy compounds

There are at least 5 groups of high energy compound.ds (table 2)

Table 2 high energy compounds

Class Pyrophosphates Acyl phosphates Bond CPP O CO~P CH CO~P O CO~S | N~ P Example (s) ATP, pyrophosphate 1,3- Bisphosphoglycerate, Carbamoyl phosphate, Acetyl phosphate. Phosphoenol pyruvate

Enol phosphates

Thiol esters (thioesters)

Acetyl CoA, Acyl CoA

Guanido phosphates (phosphagens)

Phosphocreatine, Phosphoarginine

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High energy bonds: The high energy compounds possess Acid anhydride bonds (mostly phosphoanhydride bonds) which are formed by the condensation of two acidic groups or related compounds. These bonds are referred to as high energy bonds, since the free energy is liberated when these bonds are hydrolyzed. Lipmann suggested use of the symbol ~ to represent high energy bond. For instance, ATP is written as AMP ~ P ~ P. The formation of this high energy bonds require energy and such reactions are called endergonic since energy is used. When such bonds are broken energy is given out and such reactions are known as exergonic reactions. ATP the most important high energy compound
Adenosine triphosphate (ATP) is a unique and the most important high energy molecule in the living cells. The ATP molecule is a purine (adenine) nucleotide in which the adenine is attached in a glycosidic linkage to D ribose. Three phosphoryl groups esterified to the 5 position of the ribose moiety in phosphoanhydride bonds. The two terminal phosphoryl groups (i.e., and ) are involved in the phosphoric acid anhydride bonding and are designated as energy rich or high energy bonds

ATP is found in all cells and there is every reason to believe that it is the universal supplier of energy in all biological systems, plants and animals alike.

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