Metabolic Pathways and Enzymes Cellular reactions are usually part of a metabolic pathway, a series of linked reactions Many reactions have molecules in common Energy can be released in small amounts rather than all at once Illustrated as follows: E1 E2 E3 E4 E5 E6 A → B → C → D → E → F → G Letters A-F are reactants or substrates, B-G are the products in the various reactions, and E1-E6 are enzymes. http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter8/animations.html

Enzyme - a protein molecule that functions as an organic catalyst to speed a chemical reaction. An enzyme brings together particular molecules and causes them to react. The reactants in an enzymatic reaction are called the substrates for that enzyme. For series of reactions below, A is substrate for E1 and B is product. B then becomes substrate for E2 and C is product. Continues to end of pathway. E1 E2 E3 E4 E5 E6 A → B → C → D → E → F → G

Energy of activation (Ea) - the energy that must be added to cause molecules to react with one another Enzyme lowers the amount of energy required for reaction to occur Enzymes allow reactions to take place at lower temperatures – otherwise, reactions would not be able to occur at normal body temperatures

Energy of activation (Ea) When no enzyme is present – more energy required When an enzyme is added – less energy required

Enzyme-Substrate Complexes Every reaction in a cell requires a specific enzyme. Enzymes are named for their substrates: Substrate Enzyme Lipid Lipase Ureas Urease Maltose Maltase Ribonucleic acid Ribonuclease

Active site – part of enzyme that attaches to substrate Active site may undergo a slight change in shape in order to accommodate the substrate(s) The enzyme and substrate form an enzyme-substrate complex during the reaction. The enzyme is not changed by the reaction (active site returns to its original state), and it is free to act again. http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/animation__how_enzymes_work.html

Enzymatic reaction Substrates are combined into a larger product Substrate is broken down into smaller products Substrates are combined into a larger product

Induced fit model Because the enzyme must undergo a slight change in shape to fit with the substrate, this is known as the induced fit model.

Factors Affecting Enzymatic Speed Substrate concentration Temperature and pH Enzyme concentration Enzyme inhibition Competitive inhibitors Non-competitive inhibitors Enzyme co-factors

Substrate concentration: Enzyme activity increases as substrate concentration increases because there are more collisions between substrate molecules and the enzyme. When active sites on enzymes are filled almost continuously with substrate, rate of activity cannot increase further.

Temperature and pH: As the temperature rises, enzyme activity increases because more collisions occur between enzyme and substrate. If the temperature is too high, enzyme activity levels out and then declines rapidly because the enzyme is denatured. When enzyme is denatured, its shape changes and it can no longer bind to substrate. Each enzyme has an optimal pH and temperature at which the rate of reaction is highest.

Rate of an enzymatic reaction as a function of temperature and pH

Enzyme Concentration: A cell regulates which enzymes are present or active at any one time and the quantity of enzyme present by turning on of off genes Another way to control enzyme activity is to activate or deactivate the enzyme, such as through phosphorylation (removal of phosphate group).

Enzyme Inhibition: Occurs when an active enzyme is prevented from combining with its substrate. When the product of a metabolic pathway is in abundance, it binds competitively with the enzyme’s active site, a simple form of feedback inhibition. Other metabolic pathways are regulated by the end product binding to an allosteric site (another area of enzyme). Poisons such as cyanide are often enzyme inhibitors; penicillin is an enzyme inhibitor for bacteria.

Feedback inhibition

When there is a sufficient amount of the end product, some of the product binds to the allosteric site on the enzyme, the active site changes shape, the reactant cannot bind, and the end product is no longer produced. http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter8/animations.html

Competitive inhibitors: Have a similar shape to the substrate & fit into the active site of the enzyme Don’t take part in the reaction Block active site so substrate can’t enter http://www.edumedia-sciences.com/a462_l2-competitive-inhibition.html Non-competitive inhibitors: Do not have the same shape as the substrate & do not compete for the active site Bind at some other point on the enzyme molecule, which still changes the shape of the active site so enzyme-substrate complex cannot be formed. http://www.stolaf.edu/people/giannini/flashanimat/enzymes/allosteric.swf

Enzyme Cofactors Presence of enzyme cofactors may be necessary for some enzymes to carry out their functions. Inorganic metal ions, such as copper, zinc, or iron function as cofactors for certain enzymes. Organic molecules, termed coenzymes, must be present for other enzymes to function. Some coenzymes are vitamins; certain vitamin deficiencies result in a lack of certain enzymatic reactions.

The ATP cycle

ATP (adenosine triphosphate) The energy currency of cells. A nucleotide made of the following: Adenine Ribose (a sugar) Three phosphate groups Constantly regenerated from ADP (adenosine diphosphate) after energy is expended by the cell. Pneumonic devices – ATP – a triple phosphate - ADP – a double phosphate http://www.stolaf.edu/people/giannini/flashanimat/metabolism/atpsyn2.swf

Advantages of ATP: 1) It can be used in many types of reactions. 2) When ATP → ADP + P, energy released is sufficient for cellular needs and little energy is wasted. 3) ATP is coupled to endergonic reactions (requires an input of energy) in such a way that it minimizes energy loss.

Overview of Cellular Respiration Makes ATP molecules Releases energy in several reactions Glycolysis Transition reaction Citric acid cycle (Kreb’s cycle) Electron transport system An aerobic process that requires O2

It is an oxidation-reduction reaction, or redox reaction for short. Oxidation is the loss of electrons; hydrogen atoms are removed from glucose. Reduction is the gain of electrons; oxygen atoms gain electrons. Remember OIL RIG (oxidation is loss, reduction is gain)

Enzymes involved: NAD+ Nicotinamide adenine dinucleotide Accepts 2 electrons & 1 H+ to become NADH FAD Flavin adenine dinucleotide (sometimes used instead of NAD+) Accepts 2 electrons & 2 H+ to become FADH2

The NAD+ cycle

Phases of Cellular Respiration Four phases: Glycolysis Transition reaction Citric acid cycle (Kreb’s cycle) Electron transport system (If oxygen is not available, fermentation occurs in the cytoplasm instead of proceeding to cellular respiration.)

The four phases of complete glucose breakdown

Occurs in the cytoplasm (outside the mitochondria) Glycolysis Occurs in the cytoplasm (outside the mitochondria) Glucose  2 pyruvate molecules. Universally found in all organisms Does not require oxygen. http://www.science.smith.edu/departments/Biology/Bio231/glycolysis.html

Energy-Investment Steps Energy-Investment Steps Requires 2 ATP to start process and activate glucose Glucose splits into two C3 molecules (PGAL) Each C3 molecule undergoes the same series of reactions.

Energy-Harvesting Steps PGAL is oxidized by the removal of electrons by NAD+; phosphate group is attached to each PGAL as well (phosphorylation) Removal of phosphate from 2 PGAP by 2 ADP produces 2 ATP, and 2 PGA molecules

Removal of water results in 2 PEP molecules Removal of phosphate from 2 PEP by 2 ADP produces 2 ATP molecules and 2 pyruvate molecules

Glycolysis summary Inputs: Outputs: Glucose 2 NAD+ 2 ATP 4 ADP + 2 P Inputs: Glucose 2 NAD+ 2 ATP 4 ADP + 2 P Outputs: 2 pyruvate 2 NADH 2 ADP 2 ATP (net gain) When oxygen is available, pyruvate enters the mitochondria, where it is further broken down If oxygen is not available, fermentation occurs

Inside the Mitochondria Inside the Mitochondria Structure of mitochondia: Has a double membrane, with an intermembrane space between the two layers. Cristae are folds of inner membrane The matrix, the innermost compartment, which is filled with a gel-like fluid. The transition reaction and citric acid cycle occur in the matrix; the electron transport system is located in the cristae.

Mitochondrion structure and function

Transition Reaction Is the transition between glycolysis and the citric acid cycle. Pyruvate (made during glycolysis) is converted to acetyl CoA, and CO2 is released NAD+ is converted to NADH + H+ The transition reaction occurs twice per glucose molecule.

Transition reaction inputs and outputs per glucose molecule Inputs: 2 pyruvate 2 NAD+ Outputs: 2 acetyl groups 2 CO2 2 NADH http://www.science.smith.edu/departments/Biology/Bio231/krebs.html

Citric Acid Cycle (aka Kreb’s Cycle) Occurs in the matrix of the mitochondria. C2 acetyl group (produced during transition reaction) joins a C4 molecule, and C6 citrate results. Each acetyl group gives off 2 CO2 molecules. NAD+ accepts electrons in three sites and FAD accepts electrons once. Substrate-level phosphorylation results in a gain of one ATP per every turn of the cycle; it turns twice per glucose, so a net of 2 ATP are produced. The citric acid cycle produces four CO2 per molecule of glucose.

Citric acid cycle

Citric acid cycle inputs and outputs per glucose molecule Inputs: 2 acetyl groups 6 NAD+ 2 FAD 2 ADP + 2 P Outputs: 4 CO2 6 NADH 2 FADH2 2 ATP

Electron Transport System (ETS) Electron Transport System (ETS) Located in the cristae of mitochondria Series of protein carriers pass electrons from one to the other. NADH and FADH2 carry electrons picked up during glycolysis, transition reaction, & citric acid cycle NADH and FADH2 enter the ETS.

As a pair of electrons is passed from carrier to carrier, energy is released and is used to form ATP molecules by oxidative phosphorylation (term used to describe production of ATP as a result of energy released by ETS). Oxygen receives electrons at the end of the ETS, which combines with hydrogen to form water: ½ O2 + 2 e- + 2 H+ → H2O

Overview of the electron transport system

Organization of Cristae The ETS consists of 3 protein complexes and 2 mobile carriers. Mobile carriers transport electrons between the complexes. Energy is released by electrons as they move down carriers H+ are pumped from the matrix into the intermembrane space of the mitochondrion. Produces a very strong electrochemical gradient - few H+ in the matrix and many H+ in the intermembrane space.

The cristae also contain an ATP synthase complex Hydrogen ions flow through ATP synthase complex down their gradient from the intermembrane space into the matrix. Flow of 3 H+ through ATP synthase complex causes the ATP synthase to synthesize ATP from ADP + P. This process of making ATP is called chemiosmosis, because ATP production is tied to an electrochemical gradient (H+ gradient) Once formed, ATP molecules are transported out of the mitochondrial matrix.

http://vcell.ndsu.nodak.edu/animations/atpgradient/movie.htm http://www.sp.uconn.edu/%7Eterry/images/movs/synthase.mov http://www.science.smith.edu/departments/Biology/Bio231/etc.html http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter9/animations.html

Energy Yield from Glucose Metabolism Energy Yield from Glucose Metabolism Per glucose molecule: 10 NADH take electrons to the ETS  3 ATP from each 2 FADH2 take electrons to the ETS  2 ATP from each Electrons carried by NADH produced during glycolysis are shuttled to the electron transport chain by an organic molecule (mechanism of delivery may vary # of ATP produced by ETS).

Accounting of energy yield per glucose molecule breakdown

Fermentation Occurs when oxygen is not available. During fermentation, the pyruvate formed by glycolysis is reduced to alcohol and CO2, or one of several organic acids, such as lactate. Fermentation uses NADH and regenerates NAD+, which are free to pick up more electrons during early steps of glycolysis; this keeps glycolysis going. Occurs in anaerobic bacteria, fungus, & human muscle cells. http://instruct1.cit.cornell.edu/Courses/biomi290/MOVIES/GLYCOLYSIS.HTML

Fermentation Before fermentation, glycolysis produces 2 pyruvate molecules. Then pyruvate is reduced by NADH into lactate or alcohol & CO2.

Advantages and Disadvantages of Fermentation Fermentation can provide a rapid burst of ATP in muscle cells, even when oxygen is in limited supply. Lactate, however, is toxic to cells. Initially, blood carries away lactate as it forms; eventually lactate builds up, lowering cell pH, and causing muscles to fatigue. Oxygen debt occurs, and the liver must reconvert lactate to pyruvate.

Efficiency of Fermentation Two ATP produced during fermentation are equivalent to 14.6 kcal; complete oxidation of glucose to CO2 and H2O represents a yield of 686 kcal per molecule of glucose. Thus, fermentation is only 2.1% efficient compared to cellular respiration (which is 39% efficient). (14.6/686) x 100 = 2.1%

Glycolysis and Fermentation inputs and outputs per glucose molecule Inputs (into glycolysis): Glucose 2 ATP 4 ADP + 2 P Outputs: 2 lactate (fermentation) or 2 alcohol & 2 CO2 (fermentation) 2 ADP (glycolysis) 2 ATP (net gain) (glycolysis)