Enzymes: Catalysis and Inhibition

• Enzymes catalyze biological reactions by lowering activation energy.
• Enzymes are reusable after the reaction.
• Substrates are reactants that become products.
• The active site is where the substrate binds to the enzyme.
• The active site is not a binding site.
• Induced fit models describe when the active site does not completely fit the substrate, and both the active site and substrate change shape to fit.
• Lock and key models describe when the active site fits the enzyme completely.
• Enzyme regulation occurs via inhibitors, which reduce the rate of reaction or terminate it.
• Inhibitors are normally reversible.

Competitive Inhibition

• Competitive inhibition involves inhibitors binding to the active site
• Competitive inhibition competes with the substrate for binding to the enzyme.
• Competitive inhibition leads to a decrease in the rate of reaction.

Non-Competitive Inhibition

• Non-competitive inhibition involves inhibitors not binding to the active site (allosteric site)
• This causes a change to the 3D shape of the enzyme, affecting the active site and preventing the enzyme from functioning effectively.
• Non-competitive inhibiton does not necessarily prevent substrate binding.
• Non-competitive Inhibitnon decreases the amount of product made.
• Denaturation occurs when enzymes are outside of optimal conditions, which changes its 3D shape and loses its active site shape, stopping functionality.
• Denaturation can be caused by non-optimal acidity or too high temperature, but rarely too low temperature.
• At times of denaturation, successful collisions can still occur.
• Co-enzymes cycle form loaded to unloaded carrying electrons/hydrogen ions through a biochemical reaction (e.g., ATP, NAD+, FAD+).
• Co-enzymes assist in catalyzing biochemical reactions.
• Biochemical reactions are a series of biochemical reactions controlled by enzymes.

Intro to Photosynthesis

• The purpose of photosynthesis is to convert light energy to chemical energy.
• The word equation for photosynthesis is: Water + Carbon dioxide --(Chlorophyll & Light energy)--> Oxygen + Glucose.
• Coenzymes involved in photosynthesis are NADP+ -> NADPH and ADP+Pi -> ATP.
• Chloroplasts are specialized organelles where photosynthesis occurs, found in plant cells and other phototrophs.

Chloroplast Internal Structure

Granum (Grana)

• Grana is the site of the light-dependent reaction using thylakoid membranes
• Chlorophyll is contained here and absorbs light.
• Grana have high SA:V ratios.

Stroma

• Stroma is the site of the light-independent reaction (Calvin cycle).
• This is a fluid-filled space in mesophyll or bundle sheath cells.
• Light-dependent reactions involve light energy absorbed by chlorophyll within grana of mesophyll cells
• Water is split into H+ ions and oxygen and free electrons
• NADP+ picks up H+ to form NADPH; ADP + Pi picks up e- to form ATP.
• Oxygen diffuses out of grana, then the chloroplast, then the leaf, and lastly, the stomata.
• Light-independent reactions involve CO2 diffusing through the stomata of the leaf.
• CO2 diffuse into mesophyll cells and moves into the chloroplast into the stroma.
• ATP and NADPH from light-dependent reactions fuel the Calvin cycle (carbon fixation) in the stroma.
• Glucose is produced after Calvin cycles
• Unloaded carriers (ADP + Pi & NADP+) return to the light-dependent stage.
• Limiting factors of the rate of photosynthesis: light intensity, temperature, water availability, CO2 concentration, and wavelengths of light absorbed.
• Green light is reflected, while red and blue light are absorbed best.
• White light produces the highest rate with all wavelengths.
• For plant types, C3 is limited by CO2 concentration, CAM is limited by time of day.

C3, C4, and CAM Photosynthesis

RuBisCO

• RuBisCO catalyzes the reaction between carbon dioxide and RuBP (resulting in carbon fixation) in the first stage of the Calvin Cycle.
• In high oxygen conditions, RuBisCO catalyzes a reaction between oxygen and RuBP, resulting in photorespiration.
• RuBisCO has a higher affinity to oxygen in high-heat environments.
• As an enzyme, RuBisCO has a relatively slow reaction rate.
• Enzyme activity is limited by light availability, carbon dioxide concentration, environmental temperatures, and pH levels.
• There is is potential to genetically modify RuBisCO to improve photosynthetic yields.
• Photorespiration happens when the concentration of oxygen in the leaf is higher than the concentration of carbon dioxide.
• Glucose cannot be created when photorespiration occurs and results in the use (waste) of ATP.

C3 Plants

• "Normal" plants
• Approximately 85% of plants are C3, examples include rice, wheat, soybeans & all trees
• C3 plants do not have photosynthetic adaptations to reduce photorespiration.
• C3 plants thrive in temperate environments (low temperatures, available water).
• Photosynthesis occurs when the stomata are open, and CO2 enters the C3 pathway and is immediately fixed by RuBisCO.
• In low CO2 and high oxygen conditions, C3 plants will undergo photorespiration.
• With stomata closed, there is more oxygen released in the mesophyll cell in the light-dependent reaction at high light intensity.
• With stomata closed at high temperatures, more oxygen dissolves in the cell and binds to RuBisCO at a higher rate.

Carbon Fixation in C4 and CAM Plants

• C4 and CAM plants can reduce photorespiration by fixing carbon dioxide from the environment into malate.
• The malate can be stored or moved to a different location and converted back into CO2 for use in the Calvin Cycle.
• A different enzyme (Pep-carboxylase) is used to convert CO2 into malate.
• C4 and CAM plants try to maintain a high concentration of CO2 to minimize the interaction between oxygen and RuBisCO
• CAM pathways still minimize photorespiration concentration differences.

C4 Plants

• Approximately 3% of vascular plants.
• Habitat includes: high temperatures, high humidity, and high daytime light intensity (tropical).
• The light-dependent reaction and the Calvin cycle are physically separated in different cells.
• The light-dependent reaction occurs in the mesophyll cells, while the Calvin cycle occurs in the bundle sheath cells.
• Separating these stages minimizes photorespiration due to the low oxygen concentration in the bundle sheath cells.
• CO2 is first fixed into malate and transported to bundle sheath cells.

CAM Plants

• CAM Plants are in plants adapted to arid conditions (high daytime temperatures, intense sunlight, and environments with low soil moisture).
• A Cam plant example is cacti and succulents.
• This pathway minimizes photorespiration.
• The initial carbon fixation stage is separated at night
• LDS and LIS (Calvin Cycle) occur during the day.
• Carbon is fixed into malate overnight.

CAM Plants at Night

• CAM plants open their stomata at night.
• Allows CO2 to diffuse into the leaves (O2 released).
• They convert atmospheric CO2 into malate or another organic acid and store them in vacuoles.

CAM Plants during the Day

• Light-dependent reaction (LDR) occurs at the same time (as needed coenzymes)
• Stomata are closed.
• Malate from vacuoles is converted back into CO2, which enters the Calvin cycle (light independent stage) and RuBisCO can catalyze it.
• Minimizes photorespiration as low O2 concentration.
• Very water efficient but slow process.

Aerobic Respiration

• Cellular respiration releases a usable form of energy (ATP) from glucose
• Some energy is converted to heat energy, meaning the energy transformation is not 100% efficient.
• Equation: C6H12O6 + 6O2 -> 6H2O + ATP.

Stages of Aerobic Respiration

1. Glycolysis (Cell cytosol) Afterwards oxygen is needed, and then it enters:
2. Krebs Cycle (matrix of mitochondria)
3. Electron Transport (Cristae of mitochondria)

• Mitochondria is the site of the second and third stages of areobic cellular respiration

Mitochondria Structure

• Matrix is fluid-filled spaces inside the inner membrane.
• Cristae is folded inner membrane, high SA:V ratio optimal for reactions to occur.
• In Electron Transport Chain (ETC), hydrogen ions and associated electrons are passed along a series of molecules/proteins called cytochromes which moves them along throughout the cristae.
• The process requires high Surface Area:Volume (SA:V).
• Hydrogen ions from the ETC bind with oxygen to form water, with oxygen being the final electron accepter
• There is an imbalance of hydrogen ions that fuels the reaction to produce ATP.
• Theoretical 36 ATP is not accurate since energy is lost in transport to mitochondria.

Anaerobic Respiration (Fermentation)

• Occurs in the absence of oxygen or low oxygen concentration after glycolysis.
• Glycolysis occurs in the cytosol and pyruvate is produced and then converted to different end products.
• Produces only 2 ATP for each glucose, which is less efficient, but faster than aerobic respiration.

Lactic Acid Fermentation

• Location: Cytosol
• In animal tissue, the end product is lactate.
• Non-sustainable and only occurs in bursts due to lactate buildup, which is toxic to animal tissues.
• Result: Glucose -> lactic acid + 2 ATP.

Ethanol Fermentation

• Occurs in the cytosol of bacteria, yeast, & plants
• Ethanol fermentation can continue as long as concentration isn't toxic.
• Results in Glucose -> Ethanol + CO2 + 2ATP

Biofuels

• Biofuels are renewable fuels derived from biomass (any matter usually derived from plants or animals).
• Biofuels are normally produced using bacteria and yeast in anaerobic conditions (ethanol fermentation).
• Liquid biofuels are researched and used to combat climate change and reduce greenhouse gas (CO2) emissions.
• Biomass is any organic matter from a living thing that can be used as a fuel source.
• Feedstocks are raw materials used to produce biofuel (generally carbohydrate-based from plants, e.g., cellulose or starch).
• Feedstocks can come from food crops, agricultural waste, and food waste.
• Biodiesels are fuels made from fats/oils.

Biofuel Manufacturing

1. Harvest feedstock (crop/waste).
2. Pre-treat feedstock (break down tough material to make it more accessible to enzymes using pressure, heat, mechanical breakdown to create a slurry).
3. Saccharification uses enzymes to break down cellulose/starch into monosaccharides (glucose, fructose, or sucrose) in a specific temperature and acidity. Example: Corn feedstock- Amylase is added to the liquified corn which must be conducted at a pH 5.9-6.2 and a temperature of 85-95C. Glucoamylase is added and conducted at a pH of 4.5 and a temperature of 55-65C.
4. Anaerobic fermentation follows producing glucose (produced during saccharification) which converted by yeast/bacteria into ethanol by anaerobic respiration (fermentation).
5. Purification, products is distilled to remove any unprocessed mass.

Limiting Factors of Rate of Cellular Respiration

1. Temperature: as temperature increases, particles (reactants) have more kinetic energy to result in more collisions, therefore, the rate of reaction will increase. Once the reaction is beyond the optimal temperature, there will be a rapid decrease in the rate of cellular respiration as enzymes denature.
2. Glucose availability: if there more glucose then there will be a faster rate of reaction up to a point where it will plateau (limited by other factors such as oxygen or enzyme).
3. Oxygen Concentration: the rate of aerobic cellular respiration will increase up to a point when plateau occurs (other limiting factors such as glucose, enzyme, etc).

Aerobic vs Anaerobic

Anerobic Respiration

• Oxygen not required
• Rapid ATP production
• Mammals sustain over a short time only (due to lactic acid build up)
• Plants, bacteria and yeast can sustain continuously as long as ethanol concentration doesn't become toxic.
• Less efficient energy transfer.
• 2 ATP produced per glucose used
• Various end products: lactate/lactic acid (humans) or ethanol and CO2 (yeasts, plants, bacteria)

Aerobic Respiration

• Oxygen required

• Slower rate of ATP production

• Continuous process as long as glucose is available (no toxic products)

• More efficient energy transfer

• 30,31 or 32 ATP produced per glucose used

• End products are: CO₂ and water.

• Both processes can be occurring simultaneously dependent on the concentration of oxygen contained within the cell/organism.