Chapter 3 - Cell Metabolism


Types of Metabolic Reactions
General Concepts
   Chemical reactions involve the conversion of reactants (A + B) into products (C + D). The reaction can go in either direction so that the products (C + D) become the reactants and the reactants (A + B) the products.
   By convention the reactants are written on the left side and the products on the right. A + B  C + D.  However, as already noted, the reaction can go in either direction. If more products (C + D) are being created the reaction is said to be going forward. If more reactants (A + B) are being produced the reaction is going in reverse. Forward and reverse therefore refers to the net reaction of the resulting sum of all individual reactions.
Reactions can either be:
Catabolic - When the products are smaller molecules than the reactants. e.g. proteins   amino acids. 
Anabolic - When the products are larger molecules than the reactants. e.g. amino acids   proteins  
Metabolic Pathway
   Chemical reactions normally occur in a series of stepwise reactions with the final product(s) referred to as end products and the substances in between called intermediates. This chain of reactions can be represented symbolically in the following ways:

A + X  B  C  D + Y

A   B   C   D

   In this metabolic pathway B + C are intermediates and D + Y end products.

Specific Types of Metabolic Reactions
1. Hydrolysis and Condensation Reactions
   Hydrolysis (splitting with water)(hydro -water; lysis - splitting) involves the breaking of covalent bonds with water molecules. This can be represented as:
AB + H2O
   Note that the bond between A and B is broken by attaching the hydroxyl group to A and the H to B.
   Condensation - The reverse of hydrolysis. The reaction above can go in reverse with A- OH reacting with B-H to form a larger molecule B-A with the release of H2O.
AB + H2O
2. Phosphorylation and Dephosphorylation Reactions
   Phosphorylation involves the addition of a phosphate group HPO4-2 or H2PO4-1 to a molecule. 
A + Pi
   Dephosphorylation results in the removal of a phosphate group from a molecule.
A + Pi
3. Oxidation-Reduction Reaction
   Oxidation involves the removal of electrons from any molecule. Reduction involves the addition of electrons. The two occur together in oxidation-reduction reactions with the molecule losing electrons being oxidized and the molecule gaining electrons being reduced. 
   The terms oxidation and reduction can have more subtle meanings:
1. Oxidation can refer to the reaction of a molecule with molecular (O2) even though oxygen may actually form a covalent bond (sharing electrons). As a result of the reaction the oxygen nucleus attracts the electrons more strongly and in this sense "removes electrons".  e.g. oxidation of glucose
C6H12O6 + 6O2 6CO2 + 6H2O
2. Oxidation can also refer to the removal of hydrogen atoms. Although a hydrogen atom is removed it carries with it an electron and this lose of a hydrogen atom from a molecule is considered equivalent to the lose of an electron. e.g. 
HA-BH A-B + 2H
Conversely, The addition of hydrogen is considered a reduction.

    The actual addition or removal of electrons changes the molecules electrical charge. This occurs during oxidative phosphorylation

   with the oxidation of hydrogen atoms:
2H+ + 2e-
   and reduction of hydrogen atoms:
2H+ + 2e-


Metabolic Reactions and Energy
   Metabolic reactions often involve the production or use of energy. Generally, reactions that release energy are catabolic and reactions that require energy are anabolic.
Energy Changes in Reactions
   Because molecules possess energy and different molecules possess different amounts of energy,  reactions in which molecules change from  one kind of molecule to another involve the release or use of energy. Hence, if the reactant molecules possess more energy than the product molecules energy is released: 
Reactants Products + Energy
   The energy can take various forms (heat, light, movement) and can be harnessed to perform work.
   The energy released in the above reaction can also be expressed as the change of energy between the reactants and product molecules:
DE = Eproducts - Ereactants
   If the reactant molecules have more energy than the product molecules DE  (delta energy) is negative and energy is released. 
   If the reaction requires energy:
Reactants + Energy Products
   DE is positive.
   DE is expressed in units of energy, calories (cal) or kilocalories (kcal), or in joules (J) or kilojoules (kJ). 
   The amount of energy expended or released in a reaction is proportional to the quantity of reactants involved. Hence,  DE is commonly expressed as kcal/mole.        


Types of Energy
Kinetic Energy (Energy of motion (kinesis))
   This energy is due to an objects motion. It is related to an objects mass and how fast it is moving. The kinetic energy associated with the movement of molecules is their thermal energy or heat.
Potential Energy
   Energy that has the potential to become kinetic energy. Hence, it is stored energy. e.g. Putting tension on the string of a bow creates potential energy that can be released to provide kinetic energy that impels the flight of an arrow.


    Energy releasing reactions always go spontaneously in the forward direction. Energy requiring reactions only go forward when energy is put into them. Therefore, the direction of a reaction depends upon DE.
Catabolic reactions are energy releasing and should proceed spontaneously.
Anabolic reactiosn are energy requiring and don't proceed without the input of energy.
   In cells the energy required for anabolic reactions can be gotten from the energy released by catabolic reactions if the reactions can be coupled.
   When the quantity of reactants and products in a reaction do not change the reaction is in equilibrium. In other words, there is no net reaction direction, and DE is zero.


Law of Mass Action
   The direction of a reation can be changed by altering the relative concentrations of the reactants and products. A reaction can be pushed forward by increasing the concentration of the reactants. Likewise, a reaction can be reversed by increasing the concentration of the products.


Activation Energy
   The conversion of one molecule to another is not abrupt but involves a transition state in which  the reactant assumes an intermediate form. The transition state possesses a higher potential energy than either the reactants or products and forms an activation energy barrier.
   The difference between the potential energy of either the reactants or products and the potential energy of the transition state is called the activation energy. In other words, for a reaction to go forward or in reverse additionaly energy is needed beyond the DE associated with the reaction. An additional "hump" the reaction needs to surmount. The extra energy required for the activation energy may come from the thermal movement of the molecules.


Reaction Rates
   The rate of a reaction can be expressed as a change in the concentration of the reactant molecules over time. The rate of biological reactions is important because this rate needs to match the body's needs.
   Factors affecting the rates of chemical reactions.
  1. Reactant and product concentrations.
   The rate of a reaction usually refers to the net rate because the reaction is always proceeding in both directions. An increase in the concentration of either the reactants or products will increase the net reaction in the direction that will lessen the concentration. If the concentration of the reactants is increased the net reaction will increase in the forward direction.
2. Temperature.
   The rate of a reaction increases with increasing temperature and decreases with decreasing temperature.
3. Height of the Activation Barrier
   The rate of a reaction increases as the activation energy barrier decreases.


Roles of Enzymes in Chemical Reactions
   Enzymes are biomolecules (usually proteins) that act as catalysts.
Mechanism of Enzyme Action
   Enzymes act by binding with reactant molecules (referred to as substrates) and converting them into product. The reaction is represented in this way:
   The first step in which the substrate (S) binds with the enzyme (E) is the binding step and is reversible. The second step is the catalytic step and results in the substrate being converted into product (P) and the enzyme being released. The enzyme can then be reused again and again.
   This is an oversimplification of how enzymes act since enzymes usually act on more than one substrate and produce more than one product.
Substrate Specificity
    Enzymes act upon specific substrate molecules or specific types of substrate molecules. The basis of substrate specificity is that the substrate molecule has a shape that is complementary to a site on the enzyme molecule called the active site.
   The preciseness of the fit between substrate molecules and the enzyme is described by a lock and key model because of the way a key fits into a lock by its complementary shape.
   A more realistic model is called the induced-fit model. According to this model the binding of the substrate molecule to the enzyme induces in the enzyme a conformational change (a change in the molecules shape). A good analogy for this model is the way a foot induces a change of shape of a sock which is already of a size and shape to accommodate the foot. 
Cofactors and Coenzymes
   Some enzymes can function properly only when nonprotein components called cofactors are also present. Cofactors may help the enzyme to hold its normal conformation. Cofactors may be metal ions such as iron, copper and zinc. Sometimes the cofactor is a vitamin, or is derived from a vitamin (an organic molecule obtained in the diet and necessary in only trace amounts). 
   Some cofactors function as coenzymes which do not have by themselves catalytic activity but participate directly in reactions catalyzed by enzymes. A coenzyme can function with different enzymes and hence be involved in different reactions. Usually coenzymes carry particular chemical groups from one reaction to another.
    Some coenzymes include:
1. Flavin Adenine Dinucleotide (FAD)
   Derived from vitamin B12 (riboflavin). FAD is a hydrogen (electron) carrier in oxidation-reduction reactions
2. Nicotinamide Adenine Dinucleotide (NAD+)
   Derived from the vitamin B3 (niacin). NAD+ also carries electrons in oxidation-reduction reactions both directly by carrying an electron which neutralizes the positive charge and indirectly by carrying a H atom
NAD+ + 2H NADH  +  H+
3. CoenzymeA (CoA)
   CoA is derived from vitamin B5 (pantothenic acid). The coenzyme is involved in glucose oxidation and carries acetyl groups (-CH2COOH) in certain metabolic reactions. CoA becomes covalently bonded to acetyl groups to form acetylcoenzyme A. 


Factors Affecting Rates of Enzyme Catalyzed Reactions
Enzymes can only affect the rate at which a reaction occurs. Factors that affect the rate of reactions include:
1. Catalytic Rate
   Catalytic rate is a property of the enzyme itself. Strictly speaking it is a measure of how fast the enzyme can produce product per unit time if the active site is always occupied by a substrate molecule.
2. Affinity
   This is a measure of how tightly the substrate molecule binds to the active site. Higher affinities usually mean higher rates. Affinity is influenced by how well substrate molecules fit on an active site and by the existence of any attraction between the substrate molecule and the active site such as opposite electrical charges. (Fig. 3.8)
3. Enzyme Concentration
   The more enzyme molecules there are the more product molecules can be generated per unit time. 
4. Substrate Concentration
   The higher the substrate concentration the higher the reaction rate. At higher substrate concentration the active site is occupied a higher percentage of time.
   A substrate concentration increases the percent saturation. As the proportion of occupied active site increases at some point the enzyme active sites are 100% occupied and the saturation is 100%. At 100% saturation only increasing the catalytic rate of the enzyme and/or the concentration of the enzyme will increase the catalytic rate. (Fig. 3.7)
   An enzyme with a high affinity for substrate has a higher percent saturation at an particular substrate concentration.
Other factors that influence enzyme activity include temperature and pH.    


Regulation of Enzyme Activity
   The rates of metabolic reactions need to be continually adjusted to meet the bodies varying demands. Reactions controlled by enzymes can be controlled by either increasing or decreasing enzyme concentration. However, finer control of enzyme activity can be achieved by changing the activity of existing enzyme molecules.
Allosteric Regulation
   Enzyme molecules may possess a site distinct from the active site called a regulatory site. The regulatory site has an affinity for certain molecules called modulators. When the modulator molecule binds to the regulatory site it effects a change in the enzyme conformation that alters the enzyme catalytic rate or affinity of the substrate or both. This type of regulation is called allosteric (allo- other; steric - shape).
   Modulators involved in allosteric regulation can either increase (stimulate) or decrease (inhibit) enzyme activity. Enzymes can have more than one regulatory site and be affected by more than one modulator. 
Covalent Regulation
   An enzyme may change following covalent bonding of a chemical group to a site on the enzyme. Enzymes are necessary to both  form and to break these covalent bonds. An example of covalent regulation is the phosphorylation and dephosphorylation of enzyme molecules
   Protein kinase is an enzyme that catalyzes the addition of a phosphate group
E + Pi E-P
   Phosphatase is an enzyme that catalyzes dephosphorylation
E-P E + Pi
Feedback Inhibition
   Reactions are normally linked together in chains or pathways where the product of one reaction becomes the reactant of the next reaction in the pathway, and so forth. 
In the body it is important that the pathway produces a product at a controlled rate to meet the needs of the body. One way this is accomplished is by feedback inhibition. This occurs when a product downstream of a pathway regulates the enzyme of an upstream reaction. This would happen in the example above if C was an allosteric inhibition of E2 , the enzyme that produces C.
   Feedback inhibition helps to keep the reactions going at a steady rate. So, in the same example, if the concentration of C increases, C inhibits E2, which causes the concentration of C to decline. If the concentration of C decreases, the inhibition of E2 lessens, and more C is produced.
   However, feedback inhibition is a means by which the body can respond to the changing needs of the body. In this example again if the final product D is used up at a faster rate, the third reaction increases and reduces the concentration of C. This decrease in the concentration of C in turn decreases the inhibition on E2.
   Probably the most efficient way to control a pathway is to have the end product inhibit a reaction occurring upstream. This is referred to as end-product inhibition and in the same example would work well if the product D allosterically inhibited E1 that catalyzed the first reaction in the pathway.
   Less commonly a product will stimulate or activate an enzyme catalyzing a reaction downstream. This is called feedforward activation in the example above this would occur if B allosterically stimulated E3.


ATP is the Medium of Energy Exchange
   The oxidation of glucose releases 686 kcal/mole. If this energy is not captured in some way it is simply released in the form of heat. Cells harness the energy released by oxidizing glucose by using it to synthesize adenine triphosphate (ATP).  ATP is synthesized from adenosine diphosphate (ADP) and inorganic phosphate.
ADP + Pi + energy ATP
   This is an example of a condensation reaction because H2O is produced. The energy necessary to move this reaction in the forward direction is 7 kcal/mole. The phosphate group is now covalently bonded with a high energy bond. 
   The energy in the high energy bond is released when the phosphate group is split from the molecule in a reaction that involves the use of a H2O molecule. This type of reaction is called hydrolysis (hydro- water; lysis- splitting). This reaction is represented as:
ATP ADP + Pi + energy
(H2O is usually omitted from the simplified equation)
    Energy is released when this bond is broken and the bond is called a high energy phosphate bond.


Glucose Oxidation
   Glucose oxidation is the reaction that virtually all cells rely upon for their energy needs. Oxygen reacts with glucose to yield CO2 and water in a reaction that releases energy. The reaction is:
C6H12O6 + 6O2 6CO2 + 6H2O + energy
   The energy released when glucose is oxidized is done in an incremental fashion and some steps in the metabolic pathway are coupled to the synthesis  of ATP molecules. The total amount of energy released when glucose is oxidized is 686 kcal/mole. Of this 266 kcal is used to synthesize 38 ATP molecules and the remaining 420 kcal is released in the form of heat.
Stages of Glucose Oxidation
   The oxidation of glucose can be coupled to the synthesis of ATP molecules because it is divided into stages and within these stages energy in the bonds of molecules can incrementally be transferred to the energy in the high energy phosphate bonds of ATP. 


Glycolysis (glyco-sugar; lysis- splitting)
   Occurs in the cytosol in ten reactions catalyzed by 10 different enzymes. The result is that: 
1. each glucose molecule (C6) is split into two pyruvate molecules (C3);
2. during this process two ATP's are consumed and four are produced for a net gain of 2 ATP's;
3. two molecules of NAD+ are reduced to form two molecules of NADH.
   The net results:
Glucose + 2NAD+ + 2ADP + 2Pi 2pyruvate + 2NADH + 2H+ + 2ATP
(water is also involved but not shown)
   Glycolysis does not yield much energy but its product, pyruvate, enters the next stage of glucose oxidation, the Krebs cycle. Whether or not pyruvate enters the Krebs cycle depends upon the presence of O2. When O2 is not present pyruvate is converted into lactic acid. 
Conversion of Pyruvate to Lactic Acid
   Oxygen is necessary for oxidative phosphorylation. When oxygen is not present oxidative phosphorylation cannot proceed and NADH does not get oxidized. If NAD+ is not available the ATP producing steps of glycolysis are blocked and no ATP can be produced.
   This situation can be remedied for a brief period because lactate dehydrogenase oxidizes NADH by converting pyruvate to lactate in the following reaction: (Fig. 3.22)
pyruvate + NADH + H+ lactate + NAD+
   This anaerobic reaction (without O2) cannot continue for long because:
1. it produces only a small fraction (5%) of the ATP that can be produced by aerobic metabolism;
2. lactate produced by the reaction builds up and starts to increase the acidity of the body fluids.
   The situation is relieved when oxygen again becomes available and the pyruvate again enters the Krebs citric acid cycle and the reaction that created the lactate reverses: 
pyruvate + NADH + H+ lactate + NAD+


Krebs Cycle
   As implied by cycle the Krebs cycle is not a linear series of reactions with a start and end point but a closed circle of reactions with no beginning or end point. Pyruvate is produced by glycolysis in the cytoplasm and enters the matrix of mitochondria. Pyruvate does not enter the cycle directly but indirectly by means of a linking step that coverts pyruvate into acetyl-CoA with the production of CO2 and NADH + H+.
   Each molecule of glucose produces 2 pyruvates which by means of the linking reaction yields 2 acetyl-CoA that enter the Krebs cycle. As a result of the linking step and the Krebs cycle each molecule of glucose produces:
1. 6 CO2 molecules (each pyruvate produces one CO2 in the linking step and two CO2 in the cycle);
2. 2 ATP's directly (one for each pyruvate);
3. Ten reduced coenzymes (each pyruvate in one turn of the cycle produces 4 NADH and 1 FADH2 ).
   The overall reaction for the oxidation of glucose is then:
2pyruvate + 8NAD+ + 2FAD + 2ADP + 2Pi + 6H2O 6H2O + 8NADH + 8H+ + 2FADH2 + 2ATP
   When combined with glycolysis the oxidation of glucose yields 4 ATP's (2 from glycolysis and 2 from Krebs citric acid cycle) and 12 reduced coenzymes (2 NADH from glycolysis and 10 from reduced coenzymes from Krebs cycle including the linking reaction).
   All the CO2 that will be produced is produced during the Krebs cycle and the linking step. No O2 is consumed. Oxygen consumption occurs in the next stage oxidative phosphorylation.


   The reaction that involves the addition of a phosphate group (Pi) to a molecule is phosphorylation. There are two kinds of phosphorylation:
1. Substrate Phosphorylation
   Phosphate from one molecule (substrate) is transferred to another molecule:
2. Oxidative Phosphorylation
   Involves the energy released when compounds (NADH + FADH2) release their electrons (are oxidized) and the electrons are transferred along an electron transport chain. The electrons are finally given to oxygen (final oxidation). ATP results from chemiosmotic coupling which captures the energy that is released as the electrons are transported and uses it to synthesize ATP by means of ATP synthase:


Electron Transport Chain
   When the reduced coenzymes produced by the Krebs cycle (NAD, FADH2 ) are oxidized energy is released. The electron transport chain (residing in the inner membrane of the mitochondrion) is designed to convert that energy into a form that can be used to produce ATP. The electron transport consists of a set of compounds in the inner mitochondrial membrane. Most are proteins and all are designed to capture and release electrons at various energy levels. Some of the proteins are cytochromes that contain hemes, iron containing chemical groups that are also found in hemoglobin. Other proteins are iron-sulfur proteins which contain iron atoms bound to sulfur. Coenzyme Q is not a protein but a small molecule composed of hydrocarbon. 
   NADH releases its electrons to the electron transport chain. The electrons are accepted by flavin mononucleotide  (FMN) which releases it to an iron-sulfur protein which passes it on to Coenzyme Q. The electron continues along a chain from cytochrome b to another iron-sulfur protein to cytochrome c1 to cytochrome c to cytochrome a to cytochrome a3 and finally to O2 . At each step along the chain the electron goes from a higher energy level to a lower one. (Fig. 3.18)
   As electrons pass along this chain, at three complexes of these transporters the energy that is released is used to transport a H ion from the mitochondrial matrix to the intermembrane space against its concentration gradient. This creates a concentration gradient across the inner mitochondrial membrane with a high concentration of H+ in the intermembrane space compared to the mitochondrial matrix. 
   The H+ moves down their concentration gradient through an enzyme in the inner mitochondrial membrane called ATP synthase. The energy released as H+ flows down its concentration gradient is used by ATP synthase to phosphorylate ADP to ATP. This process is called chemiosmotic coupling.
   The overall reaction for oxidative phosphorylation is:
10NADH + 10H+ + 2FADH2 + 34ADP + 34Pi + 6O2 10NAD+ + 2FAD + 12H2O + 34ATP
   When all the stages of glucose oxidation are taken together the net results is:
glucose + 6O2 + 38ADP + 38Pi 6CO2 + 6H2O + 38ATP


Energy Storage and Use
   Carbohydrates are the primary energy source in the body. Only when the supply of carbohydrates becomes limited does the body turn to fats and proteins.
Glycogen Metabolism
   Glycogen is a polymer of glucose with a branching chain. It is the storage form of glucose in animal cells as starch is the storage form in plants. When glucose is present in abundance it is converted into glycogen by glycogenesis. Glycogen is stored particularly well in skeletal muscle and liver cells. When glucose availability drops glycogen can be converted back to glucose by a process called glycogenolysis. The glucose that becomes available in skeletal muscle can be used  by skeletal muscle while the glucose from liver cells is mostly released into the bloodstream for use by other cells in the body.
   Although fat and protein can be used as an energy source, glucose must be available at all times because the nervous tissue (particularly the brain) requires it. Glycogen reserves can supply it for a period of time but when glycogen reserves are depleted glucose must be synthesized from fat and protein by a process called gluconeogenesis.
   By gluconeogenesis glucose can come from three sources: (Fig. 3.24)
1. Glycerol - A component of triglycerides. Glycerol can be converted into glycerol phosphate and enter the glycolytic pathway in reverse.
2. Lactate - Lactate can be converted into pyruvate and then enter the glycolytic pathway in reverse.
3. Amino Acids - Some amino acids can be converted after conversion to pyruvate. Others can be converted to oxaloacetic acid which can then be converted to phosphoenolpyruvate which can enter the glycolytic pathway in reverse.


Fat Metabolism
   Triglycerides are the primary storage form of fats. Fats are stored in adipose tissue where they can be broken down and used when glucose runs low. Triglycerides are broken down to their components, a glycerol molecule and three fatty acids, by a process called lipolysis. Glycerol enters the glycolytic pathway and then proceeds through the Krebs cycle and oxidative phosphorylation. Fatty acids are converted into acetyl CoA's and enter the Krebs cycle.
   Lipogenesis is the process by which fatty acids can be synthesized by reversing the reactions that were used to break them down.
    Ketones are generated as a product of fat metabolism when fat is used in abundance. Ketones can serve as an alternate source of energy for nervous tissue.
   Fats are a convenient storage molecule because they yield more energy than either carbohydrates or proteins.


Protein Metabolism
   Proteins can be broken down to amino acids by a process called proteolysis. The amino acids can be converted to pyruvate, acetylCoA and intermediates of the Krebs cycle after their amino group is removed by deamination. This produces ammonia which can be converted into urea and excreted by the kidneys.
   The reactions that yield energy by converting amino acids into pyruvates, acetyl CoA, and intermediates of the Krebs cycle are reversible. This means that carbohydrates and fats can be used to synthesize amino acids. Not all amino acids can be synthesized, however, and these are required in the diet as essential amino acids.