enzymes & metabolism notes 09

HL Biology Notes for Enzymes & Metabolism

A. De Jong/TFSS 2009 1 of 25

The information in this document covers the IB syllabus for topics 3.6, 3.7, 3.8, 7.6, 8.1 and 8.2. Enzymes Enzyme: a globular protein molecule that accelerates a specific chemical reaction. Enzymes are biological catalysts.

Active Site: the region of an enzyme surface that binds the substrate during the reaction catalyzed by the enzyme.

Substrate: the reactant(s) in an enzyme-catalyzed reaction

Denaturation: a structural change in a protein that results in a loss of its biological properties. The loss of function is usually permanent.

Exergonic & Endergonic Reactions

Exergonic reactions are those in which the free energy of the final state is less than the free energy of the initial state. (-G) This represents energy that can be used to do biological work.

Endergonic reactions are those in which the free energy of the initial state is less than the free energy of the final state. (+G)

Image from http://www.biology.arizona.edu/biochemistry/problem_sets/energy_enzymes_catalysis/graphics/8ta.gif

Image from http://www.biology.arizona.edu/biochemistry/problem_sets/energy_enzymes_catalysis/graphics/16t.gif

Enzymes increase the rate of exergonic reactions:

Exergonic reactions occur spontaneously, but do not necessarily occur quickly. By lowering the activation energy of the reaction, enzymes allow more substrate to be converted into product. Enzymes increase the number of successful collisions between reactant molecules.

Image from http://www.biology.arizona.edu/biochemistry/problem_sets/energy_enzymes_catalysis/graphics/q1ta.gif



Enzyme-Substrate Specificity

Part 1 – the Lock & Key Model

Most enzymes are specific to a specific biochemical reaction. According to the lock and key model, this is due to the three-dimensional structure of the active site which is complementary to its substrate like a lock to its key.

Image from http://neurobio.mcphu.edu/GalloWeb/lock&key.gif

According to the lock and key model, the shape of the active site is so specific that it can only catalyze one reaction. Part 2 – the Induced Fit Model

Some enzymes are can catalyze several similar reactions. The induced fit model suggests that the active site can change shape to suit the particular substrate. The active site will interact with the substrate and adapt to it to make a perfect fit.

Image from http://neurobio.mcphu.edu/GalloWeb/inducedfit.gif Enzyme Activity The speed of a reaction can be measured in two ways: how fast does the substrate disappear and how fast does the product form?

Enzyme activity is affected by the following:

Concentration

Substrate: with a fixed amount of enzyme and ample cofactors present, the rate of reaction increases as substrate concentration increases, up to the point where enzyme saturation is reached (plateau)

Enzyme: with ample substrate and cofactors present, the rate of reaction increases as enzyme concentration increases (linear relationship).



http://www.colchsfc.ac.uk/biology/newsite/brian/substrate.gif http://www.colchsfc.ac.uk/biology/newsite/brian/conc.gif

Temperature: all enzymes have an optimum temperature at which their activity is highest. High temperatures denature enzymes, and low temperatures limit enzyme activity by reducing the number of successful collisions. Human enzymes function best at body temperature (37°C). Most enzymes denature above 60°C. Thermophilic archaeans have enzymes that can function at 80°C.

Image from http://www.biologycorner.com/resources/enzyme_temp_graph.gif

pH: all enzymes have an optimum pH at which their activity is highest. Stomach enzymes function best at acidic pH, but intestinal enzymes function best in slightly basic pH.

Image from http://www.biologycorner.com/resources/enzyme_ph_graph.gif



Enzyme activity is often influenced by the presence of other chemicals. Some of these can enhance an enzyme’s activity, while others inhibit the enzyme’s action.

Some enzymes require the presence of a non-protein molecule called a cofactor. A cofactor may be an inorganic ion (e.g. Ca2+, Zn2+, K+) or a small organic molecule called a coenzyme (e.g. NAD). Sometimes, the coenzyme is permanently attached to the enzyme as a prosthetic group, and other times it only attaches during the reaction, and detaches after the reaction is completed.

Enzyme inhibitors are molecules that deactivate enzyme activity, either temporarily or permanently. Reversible enzyme inhibitors are used to control enzyme activity. There is often an interaction between the substrate or end product and the enzyme controlling the reaction. Build-up of the end product or lack of substrate may serve to deactivate the enzyme.

Image adapted from http://www.biologycorner.com/resources/enzyme_inhibition.jpg

Competitive inhibitors deactivate the enzyme by binding to the active site, blocking the substrate. For example, the antibiotic Prontosil inhibits the synthesis of folic acid in bacteria by binding to the active site of the enzyme required for folic acid synthesis. Since folic acid is a coenzyme itself, the bacterium will die if it cannot make folic acid. Animal cells are not affected, because they absorb folic acid from food.

A non-competitive inhibitor deactivates the enzyme by binding to another part of the enzyme, which still allows the substrate to bind to the active site, but slows down the rate of reaction. For example, cyanide (CN-) attaches itself to the –SH groups in an enzyme. This destroys disulfide bridges, altering tertiary structure of the enzyme. This alters the shape of the active site, and disrupts cellular respiration, which relies heavily on enzymes. If this happens to a large number of cells, the organism dies.

Allosteric enzyme inhibitors block the active site altogether, and prevent its function. This may occur when a non-competitive inhibitor binds to a part of the enzyme, and causes the shape of the active site to change. The binding of end products to an allosteric site can alter the shape of allosteric enzymes. This decreases their activity. Certain products of metabolism can act as allosteric inhibitors to enzymes that occur earlier in a metabolic pathway. This regulates metabolism according to the requirements of the organism. This is a form of negative feedback. ATP acts as an allosteric inhibitor to the enzyme phosphofructokinase (PFK), which is involved in an early stage of glycolysis.

Some enzymes require the binding of an allosteric activator in order for the substrate to bind.



Image from http://www.biologycorner.com/resources/allosteric.gif

Commercial Applications of Enzymes

1. Lactase tablets are available for individuals who cannot naturally break down lactose, the primary sugar in milk. The tablets are consumed before consumption of milk or dairy products, adding the enzyme into the digestive tract. This allows for hydrolysis of lactose into glucose and galactose, which are then metabolized for energy. “Lactose-free” milk (e.g. Lactaid®) has had the enzyme lactase added to it, so that the lactose has already been broken down.

2. Pectinase is used in the extraction of fruit juices. Pectin keeps plant cells together, so pectinase helps separate the cells, making juice extraction easier. Pectinase also makes juice more clear.

3. Laundry detergents use enzymes for stain removal, rather than phosphates, which were originally used. Phosphate build-up in water results in increased algae growth (algal bloom).

4. Baby Food is “pre-digested” by the addition of proteases that convert longer protein chains into shorter, more easily digested proteins. One brand of baby formula calls these smaller proteins “comfort proteins”.

5. Meat tenderizers are enzymes that are used to make meat less tough – essentially they are being pre-digested. This also happens when marinades are used, but acids (e.g. vinegar) are used to tenderize the meat.

6. Contact lens cleaners use enzymes to remove protein deposits from the surface of the lens.

7. Organophosphorous hydrolase enzymes break down organophosphates, which are a common compound used in pesticides (harmful to the environment!). Bacteria encoding the gene for this enzyme can be directly introduced into the environment to deliver the enzyme to aid in the bioremediation process.

NOTE:

Genetically modified bacteria produce many of these enzymes. They have the genes that encode these enzymes inserted into their genomes and produce the enzyme under various conditions (depending on where in the genome they were placed, the genes can be expressed at particular times.)



Energy in Cells Redox Reactions

Oxidation: the loss of electrons from a substance

Reduction: the gain of electrons by a substance

Many chemical reactions involve the oxidation of one substance and the reduction of another. These reactions are called redox reactions, and the oxidation and reduction occur simultaneously. The electrons lost by a substance are taken up by another.

Biological redox reactions often involve hydrogen or oxygen atoms. Since a hydrogen atom is essentially a hydrogen ion plus an electron, gaining a hydrogen atom is considered to be a reduction. Losing an oxygen atom is also a reduction. Conversely, losing a hydrogen atom is an oxidation, as is gaining oxygen.

During cellular respiration, the coenzyme NAD accepts hydrogen ions and electrons, becoming reduced:

NAD+ + H+ + 2e- NADH (oxidized NAD) (reduced NAD)

NADH is oxidized when the hydrogen ions (protons) and electrons are released.

Oxidizing Agents

A substance with a strong tendency to take electrons from another substance is called an oxidizing agent. NAD+ is an oxidizing agent. Oxidizing agents become reduced as they oxidize other substances.

The net result of cellular respiration is the oxidation of glucose to carbon dioxide. The main oxidizing agent in cellular respiration is NAD+.

Reducing Agents

A substance with a strong tendency to lose electrons to other substances is called a reducing agent. NADH is a reducing agent. Reducing agents become oxidized as they reduce other substances.

The net result of photosynthesis is the reduction of carbon dioxide to carbohydrate, (CH2O)n, as hydrogen is added to carbon dioxide. The main reducing agent in photosynthesis is NADP.

Oxidation Reduction • addition of oxygen • removal of hydrogen • loss of electrons • release of energy

• removal of oxygen • addition of hydrogen • gain of electrons • uptake of energy

Energy and the Laws of Thermodynamics Energy is the ability to bring about change or do work, and exists in many forms (e.g. light, sound, heat).

• Potential energy: energy that has not been used (i.e. stored energy).

• Kinetic energy: energy that is in use (or in motion).

• Thermodynamics: the study of energy.

First Law of Thermodynamics

Energy can be changed from one form to another. It cannot be created or destroyed. The total amount of energy in the universe remains constant.



Second Law of Thermodynamics

In all energy exchanges, if no energy enters or leaves the system, the potential energy of the final state will always be less than that of the initial state. This means that there is always energy loss (as heat) when energy changes form. For example, a calculator will only run as long as there is potential energy stored in its batteries. Once the potential energy is used up, the batteries need to be replaced or recharged.

This is also known as thermodynamic entropy: For a closed system, the quantitative measure of the amount of thermal energy not available to do work.

Today it is customary to state the second law of thermodynamics as follows: Entropy in a closed system can never decrease.

Bond Energy & Free Energy For any particular chemical bond, the amount of energy it takes to break that bond is exactly the same as the amount of energy released when the bond is formed. This value is called bond energy. Since all forms of energy are eventually turned into heat, biologists tend to measure energy in kilocalories, the amount of heat required to warm one litre of water by one degree Celsius.

Free energy is energy that can be harnessed to do work. The energy stored in chemical bonds is a type of free energy. The conversion of free energy to work is never 100% efficient. Much of the energy is lost as heat, which is no longer free energy. When we use our muscles to lift an object, we are using the free energy stored in the bonds of food molecules. We use the letter G to represent free energy, and ΔG to indicate a change in free energy.

For example, the following represents the electrolysis of water into hydrogen and oxygen:

2H2O 2H2 + O2, ΔG=+118 kcal

The positive value for free energy indicates that it has been stored in the bonds of the hydrogen and oxygen molecules. In the reverse reaction, synthesis of water, ΔG = -118 kcal.

Mitochondria synthesize water using the hydrogen atoms removed from organic molecules (e.g. glucose) and the oxygen atoms they take in as they respire. This process is called cellular respiration, and its overall equation is:

C6H12O6 + 6O2 6CO2 + 6 H2O, ΔG=-686 kcal

This process occurs in several stages, and mitochondria are able to trap some of the energy of glucose in ATP.

ATP ATP, adenosine triphosphate, is a nucleotide that performs many essential roles in the cell. It is the major energy currency of the cell, providing energy for most of the energy-consuming activities of the cell. It is one of the monomers used in the synthesis of RNA, and after conversion to deoxyATP, DNA. It regulates many biochemical pathways.

A single working muscle cell consumes ~600 ATP/min and the human body consumes its own mass in ATP every day!

Image from http://www.uic.edu/classes/bios/bios100/summer2003/atp.jpg



In the diagram above, the bonds between the first and second, and second and third phosphates are considered to be “high energy”, and are indicated with a heavy line to distinguish them from the others. When the third phosphate group of ATP is removed by hydrolysis, a substantial amount of free energy is released. This also occurs when the second phosphate is removed from ADP.

ATP Production in Cells There are two methods of ATP production in cells:

1. Substrate-Level Phosphorylation involves the transfer of a phosphate from a high-energy (i.e. food) molecule to ADP with the assistance of an enzyme.

Image from http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/metabolism/energy/images/sublev_il.jpg

2. Chemiosmosis requires a phospholipid bilayer, a proton pump (intrinsic protein), protons (H+), and ATPase (ATP synthase). Energy from food molecules is used to pump protons out, creating a high concentration of protons outside the membrane. Protons come back through the membrane by facilitated diffusion through a channel in ATPase, activating the enzyme. ATPase catalyzes the formation of ATP from ADP.

Image from http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/pmf/images/u4fg33b.jpg



Image from http://home.earthlink.net/~dayvdanls/FormingATP.GIF

Glycolysis All cells use glycolysis to produce ATP:

• glucose (6 carbons) is split into two molecules of pyruvate (3 carbons each)

• net production of ATP is 2

• efficiency of the process is 2%, because there is still a lot of energy stored in the bonds of pyruvate

• anaerobic process, can run in aerobic or anaerobic conditions

• 10 reactions from start to finish

Overall, the process of glycolysis can be broken down into four stages:

1. Glucose mobilization (phosphorylation): Glucose is converted to fructose-1,6-diphosphate by substrate-level phosphorylation, with the expenditure of 2 ATP.

Net Reaction:

glucose + 2 ATP fructose-1,6-diphosphate + 2 ADP

2. Cleavage (lysis): Fructose-1,6-diphosphate is cleaved into two 3-carbon molecules. One is glyceraldehyde-phosphate (G3P). The other is converted into G3P. (G3P is also called PGAL, for phosphoglyceraldehyde.)

Net Reaction:

fructose-1,6-diphosphate 2 glyceraldehyde-3-phosphate (G3P)

3. Oxidation of G3P: Removal of a hydrogen atom carrying two electrons results in the reduction of NAD+ to NADH.

4. Production of ATP: Each molecule of G3P is converted into pyruvate (3-carbons), with a total of 4 ATP produced.

Net Reaction:

2 glyceraldehyde-3-phosphate + 2 Pi + 4 ADP 2 pyruvate + 4 ATP



The Glycolysis Pathway

Image from http://plantphys.info/principles/images/glycolysis.gif

Mitochondria

Mitochondria are self-replicating organelles found in most eukaryotic cells. They are enclosed by a double membrane, and because of this, they are thought to have originated as symbiotic aerobic bacteria which were engulfed by prehistoric cells. This theory is called the Endosymbiotic theory, and includes the chloroplasts of plant cells.



Image from http://www.mansfield.ohio-state.edu/~sabedon/campbl09.htm

Image from http://www.estrellamountain.edu/faculty/farabee/biobk/BioBookCELL2.html

The outer membrane encloses the entire structure. It is thought to have been derived from the host cell’s plasma membrane. It contains many complexes of integral membrane proteins, which serve as channels for many ions and molecules.

The inner membrane encloses a fluid-filled matrix, and is thought to have been derived from the bacterial membrane. It is folded into cristae, in a similar fashion to bacterial membranes. Folding the membrane into cristae helps to increase the surface area for the electron transport chain.

The matrix contains a mixture of enzymes that catalyze the catabolism of pyruvate and other small molecules. It is the site of the Citric Acid Cycle (a.k.a. Kreb’s Cycle).

What Happens to Pyruvate? No Oxygen?? Fermentation!

Fermentation is a form of anaerobic respiration, which occurs when there is insufficient oxygen for the Krebs cycle to occur. In animals, this may be a result of oxygen debt caused by exercise. In animal muscle tissue, pyruvate may be converted into lactic acid, by fermentation. In plants, pyruvate may be converted into ethyl alcohol, with the release of CO2. The elimination of pyruvate allows glycolysis to continue, and further production of ATP. Fermentation produces no ATP.



Fermentation Pathways

Image from http://fig.cox.miami.edu/~lfarmer/BIL265/BIL2001/fermentation.jpg

Lots of Oxygen?? Krebs Cycle!!

The citric acid cycle is often referred to as the Krebs cycle, after Sir Hans Krebs, who worked out the details of the cycle in the 1930’s.

After completion of glycolysis, pyruvate enters the mitochondria, where it is decarboxylated (CO2 is

removed). After decarboxylation, a hydrogen atom with two electrons remains, which reduces NAD+ to NADH. The other remnant is a two-carbon acetyl group. The acetyl group is added to a cofactor called coenzyme A (CoA), forming acetyl-CoA. A multi-enzyme complex catalyzes this series of reactions. The overall reaction, called the link reaction, may be represented by the following equation:

pyruvate + NAD+ + CoA acetyl-CoA + NADH + CO2

The NADH produced in this reaction is later used to produce ATP. Acetyl-CoA is important because it is produced from the breakdown of lipids and proteins, as well as glucose. This means that we can use the energy from all food molecules to make ATP, not just carbs.

Image from http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/metabolism/cellresp/images/u4fg34c.jpg



The Krebs cycle oxidizes Acetyl-CoA. The first step in this process is the binding of acetyl-CoA to oxaloacetate, a four-carbon molecule. The resulting six-carbon molecule is citrate (citric acid). Citrate is passed through a series of electron-yielding oxidation reactions, during which two CO2 are split off. This regenerates oxaloacetate, which may continue the series of reactions with another acetyl-CoA.

The nine reactions of the Krebs cycle may be grouped into two stages:

1. Preparation: Acetyl-CoA joins the cycle, and two reactions cause rearrangement of chemical groups.

2. Energy Extraction: Four of the six remaining reactions are oxidations in which electrons are removed. One generates an ATP by substrate-level phosphorylation.

Together, these reactions make up a cycle that begins and ends with oxaloacetate. For each molecule of glucose that is metabolized, the Krebs cycle runs twice, oxidizing acetyl-CoA to CO2 and H2O, releasing electrons, which will eventually drive the proton pumps that generate ATP in the electron transport chain. These electrons are temporarily housed within NADH and FADH2 molecules.

Image from http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/metabolism/cellresp/images/u4fg35.jpg

Products of the Krebs cycle:

2 ATP are produced by substrate-level phosphorylation 8 NADH are produced by oxidation 2 FADH2 are produced by oxidation

Electron Transport Chain (ETC)

The ETC puts the electrons released by the oxidation of glucose to work driving proton-pumping channels. The final acceptor of the electrons released from pyruvate is oxygen, which is needed to form oxygen.

The molecules of NADH and FADH2 formed during glycolysis and the Krebs cycle each contain a pair

of electrons gained when NAD+ was reduced to NADH, and FAD was reduced to FADH2. The NADH molecules take their electrons to the membrane, where they are transferred to an intrinsic protein complex called NADH dehydrogenase. FADH2 is attached to the inner mitochondrial membrane. The electrons from NADH are passed from NADH dehydrogenase to a series of intrinsic proteins called cytochromes and other carrier molecules, where their energy is used to drive three transmembrane proton pumps. This series of electron carriers is the electron transport chain (ETC). The electrons from FADH2 enter the ETC after those from NADH, and only activate two proton pumps. Thus, oxidation of one molecule of NADH yields three ATP, while oxidation of one molecule of FADH2 yields only two ATP.



The final step of the ETC, the cytochrome c oxidase complex, uses four electrons to reduce a molecule of oxygen gas to water. The reaction is:

O2 + 4 H+ + 4 e- 2 H2O

Since oxygen is the final acceptor of electrons in the ETC, a lack of oxygen halts the entire process. When this occurs, each acceptor molecule in the chain is stuck with its electrons until there is more oxygen available. Since most aerobic organisms cannot survive on the ATP produced by glycolysis alone, lack of oxygen causes death. Some poisons, such as cyanide, also halt the ETC, by binding to a cytochrome, inhibiting it from passing its electrons on to oxygen.

Image from http://student.ccbcmd.edu/~gkaiser/biotutorials/energy/images/chemios_il.jpg

The Interconversion of Fuels - Metabolism of Molecules Other Than Glucose The immediate source of energy for most cells is glucose. But glucose is not the only fuel on which cells depend. Cells can obtain energy from:

•other carbohydrates •fats •even proteins

These macromolecules do not require separate pathways from those used for glucose metabolism. One of the great advantages of the step-by-step oxidation of glucose into CO2 and H2O is that several of the intermediate compounds formed in the process link glucose metabolism to the metabolism of other food molecules. For example, when fats are used as fuel, the glycerol portion of the molecule is converted into G3P (PGAL) and enters the glycolytic pathway at that point. Fatty acids are converted into molecules of acetyl-CoA and enter the respiratory pathway to be oxidized in the mitochondria. The amino acids liberated by the hydrolysis of proteins can also serve as fuel:

•The nitrogen is removed in a process called deamination. •The remaining fragments then enter the respiratory pathway at several points.

For example: •the amino acids Glycine, Serine, Alanine and Cysteine are converted into pyruvate and enter the mitochondria to be respired. •acetyl-CoA and several intermediates in the Citric Acid Cycle serve as entry points for other amino acid fragments

These links thus permit the respiration of excess fats and proteins in the diet. Animals that depend largely on ingested fats (e.g., many birds) or proteins (e.g., carnivores) require no special mechanism of cellular respiration for their energy supply.



Much of the protein we consume is ultimately converted into glucose (a process called gluconeogenesis) to provide fuel for the brain and other tissues. Although all our foods are interconvertible to some extent, they are not completely so. In other words, no single food can supply all our anabolic needs. We can indeed synthesize many fats from glucose, but certain unsaturated fats cannot be synthesized and must be taken in directly in our diet. Although we can synthesize 11 of the amino acids from carbohydrate precursors, we must obtain 9 others (the "essential amino acids") directly. Many of the points that connect carbohydrate metabolism to the catabolism of fats and proteins serve as two-way valves. They provide points of entry not only for the catabolism (cellular respiration) of fatty acids, glycerol, and amino acids, but for their synthesis (anabolism) as well. Thus the catabolic breakdown of starches can lead (through acetyl-CoA and PGAL) to the synthesis of fat (as so many of us know!).

Overview of Cellular Respiration

Image from http://library.thinkquest.org/C004535/media/catabolism.gif



Photosynthesis Structure of the Chloroplast

Image from http://www.agri.huji.ac.il/~zacha/images/chloroplast.jpg

Image from http://www.daviddarling.info/images/chloroplast.jpg

Introduction to Photosynthesis

• plant are autotrophs, organisms that convert sunlight energy into chemical energy the process used is called photosynthesis, and converts carbon dioxide from the

atmosphere into carbohydrates and other organic compounds carbon dioxide + water → glucose + oxygen products of photosynthesis are used by organisms for cellular respiration (this includes

autotrophs and heterotrophs) • actually consists of many biochemical reactions, which occur in two stages:

light-dependent reactions which capture energy light-independent reactions which turn carbon dioxide into carbohydrates

• sunlight, which is perceived as white light, is actually made up of many colours (ROYGBIV), and is just a small portion of the electromagnetic (EM) spectrum

• a spectrophotometer is an instrument that is used to analyze light – specifically, the wavelength (λ) or colour being absorbed

• plants use light in the 450 nm (indigo-blue) and 700 nm (orange-red) range the reason they appear green is because green light is being reflected, not absorbed like

the blue and red • plants are able to absorb light energy because of the presence of pigments, primarily

chlorophyll a and b, located in the chloroplast



The Electromagnetic Spectrum

Image from http://www.chem.ucalgary.ca/courses/351/Carey/Ch13/1301.gif

Photosynthetic Pigments

1. Chlorophyll a • absorbs violet-blue and red light strongly • absorbs only small amounts of green, yellow and orange light • only pigment that can transfer light energy to carbon fixation reactions • “blue-green” chlorophyll

2. Chlorophyll b • absorbs blue light very strongly and violet and orange lights moderately • absorbs only small amounts of green, yellow and red light • accessory pigment, absorbing only those wavelengths that chlorophyll a cannot • “yellow-green” chlorophyll

Both chlorophylls absorb green light poorly, giving plants their characteristic green colour.

3. Carotenoids • absorb violet and blue-green light strongly • transmit shades of yellow, orange and red • energy-absorbing role protecting chlorophyll from damage (caused by absorbing a lot of

light) • e.g. β-carotene

Absorption Spectra vs. Action Spectrum Action spectra display the effectiveness of different wavelengths of light on photosynthesis, plotting rate of photosynthesis versus wavelength. The absorption and action spectra are similar for chlorophyll a but are not identical due to the presence of accessory pigments. Engelmann's Experiment

• Engelmann used a prism to illuminate a filament of algae with different wavelengths of light • added aerobic bacteria (attracted to O2) to detect regions producing the most oxygen and

therefore would have the highest rates of photosynthesis • bacteria congregated where violet-blue or red light was illuminated



Image from http://www.ualr.edu/botany/spectra.gif Photosystems Photosystems are protein complexes involved in photosynthesis. They are located in the thylakoid membranes of the chloroplast. Photosystems use light to reduce molecules. Light energy is absorbed by a reaction centre, and used to excite electrons, which are passed through an electron transport system, eventually reducing NADP+.



Image from http://www.ualr.edu/botany/chlorophyll.jpg The Light-Dependent Reactions

• convert solar energy to chemical energy • requires two photosystems (PS) and an electron transport system in the thylakoid membrane • PSII a.k.a. P680 absorbs light at an average wavelength of 680 nm (red) • PSI a.k.a. P700 absorbs light at an average wavelength of 700 nm (far red) • water molecules are split, producing oxygen (waste product), electrons (e-) and protons (H+)

leading to the formation of NADPH this process is called photolysis (splitting using light energy)

• chemiosmosis also drives production of ATP (photophosphorylation) • NADPH and ATP are both required by the light-independent reaction (Calvin Cycle)

Details of the Light-Dependent Reaction (Z-Pattern)

1. A photon (unit of light) strikes PSII exciting (energizing) an e- in the P680 complex (photoreceptor).

2. The primary electron acceptor captures the electron. 3. An enzyme splits a water molecule releasing 2 H+, 2 e- and O, which forms oxygen gas (O2) in

the thylakoid lumen. The electrons released here replace those lost by PSII to the primary electron acceptor. Protons build up in the thylakoid lumen, creating a concentration gradient.

4. Electrons are then passed through an electron transport chain (ETC) consisting of plastoquinone (PQ), cytochrome (cyt) and plastocyanin (PC) proteins.

5. In PSI, light excites electrons in P700 to its primary electron acceptor. The excited electron is replaced by one carried from the ETC (from PC).

6. The electron then passed through another ETC consisting of ferrodoxin (Fd), then to FAD (an NADP+ reducing enzyme) to produce NADPH.

7. Diffusion of protons back along the concentration gradient from the lumen to the stroma through the ATP synthase protein produces ATP from ADP and Pi.



8. Sometimes, electrons take an alternative path that uses PSI only. Electrons cycle back from ferrodoxin to the cytochrome complex and continue back to the PSI complex. This cycle drives translocation of protons across the membrane, further increasing the concentration gradient. This increases ATP production.

Steps 1-6 are known as non-cyclic electron flow. Step 7 is known as photophosphorylation and is driven by chemiosmosis. Step 8 is known as cyclic electron flow: flow of photon-energized electrons from PSI through

an ETC that produces ATP by chemiosmosis, but no NADPH is produced. Calvin cycle consumes more ATP than NADPH, therefore cyclic electron flow makes up the difference.

Image from http://en.wikipedia.org/wiki/Light-dependent_reaction The Light-Independent Reactions

• second stage of the photosynthetic process; takes place in the stroma • anabolic process (producing larger molecules from smaller subunits) • cyclic process: starting molecule is regenerated after molecules enter and leave the cycle • three phases: carbon fixation, reduction rxns, and regeneration of CO2 acceptor • ATP and NADPH supplied by the light dependent reactions to convert carbon dioxide into a

sugar called glyceraldehyde-3-phosphate (G3P) • discovered by Melvin Calvin's group in 1961 and resulted in the Nobel Prize • a.k.a. C3 photosynthesis (because the first stable molecule CO2 is incorporated into has three

carbons)



Image from http://student.ccbcmd.edu/~gkaiser/biotutorials/photosyn/images/u4fg46.jpg

Phase 1: Carbon Fixation

1. One CO2 at a time is attached to a 5-carbon molecule called ribulose bisphosphate (RuBP) through the action of an enzyme called ribulose bisphosphare carboxylase oxidase (RUBISCO). This reaction occurs six times (i.e. 6 CO2 molecules total are incorporated).

2. An intermediate, 6-carbon compound is formed which then breaks down into two smaller 3-carbon compounds called phosphoglycerate (PG). For each CO2, two PG molecules are produced, therefore after all six CO2 molecules have

been converted, twelve PG total are produced. Phase 2: Reduction Reactions

1. Each PG is converted to 1,3-bisphosphoglycerate (1,3-BPG) and a molecule of ATP is broken down to ADP. ATP releases energy to drive the reaction by breaking one of its phosphate bonds. An enzyme called phosphoglycerate kinase catalyzes this reaction. Kinases are enzymes

that transfer phosphate groups between different compounds. 2. 1,3-BPG is converted to glyceraldehyde-3-phosphate (G3P) and NADPH is oxidized,

producing NADP+, 2 H+ and 2 e-. A dehydrogenase enzyme catalyzes this reaction (catalyzes a transfer of a proton, H+). One phosphate group per G3P molecule is also removed. From here, two molecules of G3P exit the cycle, eventually forming glucose by reverse

glycolysis.



G3P is a key intermediate!

Used for cellular metabolism to produce glucose (in cytoplasm, then used for glycolysis). When glucose is in excess, polymerization occurs to form amylose, amylopectin, and stored as

starch (in the stroma) Glucose can be converted to sucrose in the cytosol of plant cells and exported via

translocation to other parts of the plant (via the phloem). G3P is formed after cleavage of fructose-1,6-bisphosphate in the glycolytic pathway – further

proof of the interconversion of fuels. Phase 3: Regeneration of the CO2 Acceptor, RuBP

1. Through a series of complex reactions, the ten remaining G3P (30-C total) are converted back to form six molecules of RuBP (6 x 5-C = 30-C). During this chain of reactions, six molecules of ATP are consumed to release energy to drive the reactions forward, producing six ADP molecules.

Important Notes!

The ADP and oxidized NADP+ that are formed throughout the Calvin cycle are subsequently re-used for the light dependent reactions.

The Calvin cycle occurs in both light and dark conditions, but occurs at a slower rate in the dark. It is no longer referred to as the dark reactions.

The net equation for the Calvin cycle per G3P produced is:

3CO2 + 9ATP + 6NADPH 9ADP + 8 Pi + 6 NADP+ + G3P

**this doubles when you consider that it takes two G3P to make one glucose! More ATP is required than NADPH (9:6), which is why there is cyclic and non-cyclic electron

flow in the light dependent reaction. Alternative Pathways of Carbon Fixation

• photorespiration occurs when light is present during hot, dry days in C-3 plants • heat causes stomata openings to decrease in size to prevent transpiration • CO2 absorption and [CO2] in leaves' air spaces • [O2] compared to CO2

(cellular respiration continues during this time) Effect on RUBISCO:

CO2 and O2 compete for the active site of RUBISCO [CO2]>[O2]: RUBISCO catalyzes CO2 fixation to RuBP [CO2]<[O2]: RUBISCO oxidizes RuBP

RuBP oxidation 3PG + glycolate (C5) (C3) (C2)

The 3PG produced by oxidation of RuBP continues to Calvin cycle, but the glycolate does not. It may result in the production of some CO2.



The C4 Pathway C4 plants include sugar cane, corn, and members of the grass family. C4 plants utilize an additional carbon fixation step that precedes the Calvin cycle, where CO2 is first fixed into a 4-carbon compound. This process reduced the amount of photorespiration that takes place and continuously pumps CO2 molecules back into Calvin cycle, preventing RUBISCO from binding O2. Steps:

1. PEP carboxylase converts CO2 and PEP (phosphoenolpyruvate) (C-3) to form oxaloacetate (C-4).

2. Oxaloacetate is converted to malate. **These two steps occur in the cytoplasm of a mesophyll cell.

3. The malate then diffuses into bundle sheath cells through a cell to cell connection called plasmodesmata.

4. The malate is then broken down through a decarboxylation reaction into pyruvate and CO2. 5. The pyruvate then diffuses back into the mesophyll cell and is converted back into PEP. In the

process, one ATP is dephosphorylated into ADP. 6. The CO2 undergoes a second fixation that is catalyzed by RUBISCO in the Calvin cycle.

**Note this is a more energy-consuming process, since an additional ATP is used.

Image from http://www.ualr.edu/botany/c4pathway.gif



CAM – Crassulacean Acid Metabolism Occurs in succulent (water-storing) plants, in hot, arid conditions (e.g. Cacti, pineapples). Plants open their stomata at night and close them during the day. This helps to conserve water, but also prevents CO2 from entering the leaves.

1. Night: CO2 enters leaves and it becomes fixed into organic molecules organic acids are stored in mesophyll cells in vacuoles.

2. Day: light dependent reactions supply ATP and NADPH for the Calvin cycle CO2 is released from organic acids and is incorporated into carbohydrates

Image from http://www.vcbio.science.ru.nl/images/blad/IL028_500m_nedCrassulaceanZuurMetabolisme.gif Factors Affecting the Rate of Photosynthesis Light

Image from http://ghs.gresham.k12.or.us/science/ps/sci/ibbio/cellenergy/photopics/liteintensity.gif



Temperature

Image from http://ghs.gresham.k12.or.us/science/ps/sci/ibbio/cellenergy/photopics/temprate.gif

Carbon dioxide

Image from http://ghs.gresham.k12.or.us/science/ps/sci/ibbio/cellenergy/photopics/co2rate.gif Measuring the Rate of a Reaction Reaction rates can be measured directly by observing rate of product formation, or rate of disappearance of substrate (reactant). In photosynthesis, this means either measuring the rate of oxygen gas production, or rate of carbon dioxide consumption. Photosynthesis may also be indirectly measured by the increase in biomass.

enzymes & metabolism notes 09

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