Enzyme Kinetics PDF

Review for Quiz ⬜ Passive vs active ⬜ Tonicity ⬜ Permeability factors ENZYME KINETICS Lab Outline ⬜ Define: ◼ Enzyme ◼ Energy of activation ◼ Total energy change of a reaction ⬜ Effect of temperature and pH ⬜ Enzyme activity of mLDH and its reaction Chemical Reaction Rates ⬜ The RA...

Review for Quiz ⬜ Passive vs active ⬜ Tonicity ⬜ Permeability factors ENZYME KINETICS Lab Outline ⬜ Define: ◼ Enzyme ◼ Energy of activation ◼ Total energy change of a reaction ⬜ Effect of temperature and pH ⬜ Enzyme activity of mLDH and its reaction Chemical Reaction Rates ⬜ The RATE of a chemical reaction is a measure of how fast it consumes reactants and generates products Expressed in concentration per unit time ◼ [ ] read as “concentration of” whatever is in the brackets ◼ A+B→C+D The concentrations of A and B diminish, while the concentration of C and D increase. Significance of Reaction Rate ⬜ ⬜ Reaction rates must match the body’s instantaneous demands EXAMPLE: Hypothermia Body temperature falls, decreasing rates of metabolic reactions ◼ If reaction rates slow too much…DEATH occurs ◼ Factors Affecting Rates of Chemical Reactions 1. Reactant and Product Concentrations Net Rate: difference between rate of forward reaction and reverse reaction ◼ Increased [reactants] → increased rate of forward reaction ◼ If enough product is produced, reaction can reverse ◼ ◼ Take home message: more molecules present → increased frequency of collisions between molecules → increased rate of reaction Factors Affecting Rates of Chemical Reactions 2. Temperature Increased temp: increases kinetic energy of molecules…which increases the energy of collisions ◼ Decreased temp: decreases kinetic energy of molecules…therefore decreasing collision energy ◼ ◼ Take home message: warmer temps get molecules moving faster, which means they bump into each other with more force → increased rate of reaction Factors Affecting Rates of Chemical Reactions 3. Height of Activation Energy Barrier Molecules must collide with a specific amount of force in order to react…Activation Energy ◼ Only a fraction of molecules have sufficient collision energy to react ◼ If a reaction has a lower activation energy barrier, more products can be generated ◼ ▫ Analogous to high-jump…if you lower the bar, more people can jump over it ◼ If a reaction has a higher activation energy barrier, less products can be generated Products from reaction with low AEB Products from reaction with high AEB Enzymes Increase Reaction Rate Enzyme = biological catalyst ⬜ Enzymes are substrate specific ⬜ Enzymes do not change the nature of the reaction or the final product ⬜ Enzymes themselves are not changed by the reaction ⬜ E enzyme + S E∙S P substrate enzymesubstrate complex product + E enzyme Substrate Specificity ⬜ Lock and key model: the shape of the substrate complements the active site of the enzyme *Doesn’t explain reversible reactions Substrate Specificity ⬜ Induced-fit Model: both the substrate and the product can bind to the active site, allowing the reaction to be reversible Enzymes ↓ Activation Energy ⬜ AE = the energy necessary for reaction Factors Affecting Rate of Enzyme-Catalyzed Reactions ⬜ Many things impact the rate of enzyme activity: Temperature ◼ pH ◼ Cofactors and coenzymes ◼ Concentration of enzyme and substrate ▫ Saturation ◼ Affinity ◼ Temperature Changes in temperature alter protein structure ⬜ Body Temp (~37°C) is tightly regulated, so changes in temp are rarely significant ⬜ ↑ Temp = ↑ enzyme rate…up to optimum pH ⬜ pH of intra and extracellular fluid is also very tightly regulated (7.2-7.4) ◼ Optimal pH depends on location Generally speaking, increasing acidity (decreasing pH) ↓ enzyme activity by causing structural changes as well as altering the charge at the active site Cofactors Coenzymes Vitamin-derived cofactors ⬜ Carbon based ⬜ Able to carry chemical groups from one reaction to another ⬜ Unchanged by reactions…can be reused ⬜ Important Metabolic Coenzymes: ⬜ FAD: Flavin Adenine Dinucleotide (derived from B12) ◼ NAD: nicotinamide adenine dinucleiotide (derived ◼ from B3) ◼ CoA: Coenzyme A (derived from B5) Saturation ⬜ Higher concentrations of substrate or enzyme result in faster reaction rate Affinity ⬜ Measure of how tightly substrate molecules bind to the active site of an enzyme Regulation of Enzyme Activity ⬜ ⬜ ⬜ The body regulates the activity of enzymes to adjust the rates of reactions in response to changing demands Changing enzyme concentration is time consuming Methods for altering enzyme activity: Allosteric Regulation ◼ Covalent Regulation ◼ Feedback Inhibition (end product inhibition) ◼ Allosteric Regulation Allosteric Regulation pt. 2 Covalent Regulation Feedback Inhibition The inhibited enzyme is often the rate-limiting enzyme Experimental Procedure ⬜ Effects of altering pH and temperature on the activity of lactate dehydrogenase (LDH) LDH ⬜ ⬜ Located in heart, liver, muscle, erythrocytes Conversion of pyruvate and NADH → lactate and NAD+ ◼ ⬜ Glycolysis mLDH General Experimental Protocol 37°C pH 7.4 25°C NADH pH 8.5 60°C pH 5.5 Rat Muscle Homogenate Pyruvate Spectrophotometer…(aka Spec) What are we measuring? Light absorbance by NADH ⬜ NADH absorbs light maximally at 340 nm ⬜ Light Photoelectric cell Blank – dH2O Photoelectric cell Light 0 % Absorbency reading = 0.00 100% 60 % Absorbency reading = .300 40 % 30 % Absorbency reading = .150 70 % Start – High NADH Light Later – Lower NADH Light Take Home Message ⬜ Decreased Absorbance ➔ Decreased NADH ⬜ Are we measuring mLDH activity directly? What can we predict for mLDH? ⬜ Temperatures of NADH: 25°C ◼ 37°C ◼ 60°C ◼ ⬜ pHs of NADH: 5.5 ◼ 7.4 ◼ 8.5 ◼ Time to work… Groups… Roles and Protocol Tips ⬜ 5 groups (1 per spec) ◼ ◼ ◼ ◼ ◼ ⬜ ⬜ ⬜ ⬜ 1 person zero spec before each reading 1 person to handle sample 1 person to time the reaction 1 person record absorbances 1 person to do calculations Anyone touching the cuvettes must wear gloves Invert 3 times to mix with parafilm Zero spec between each reading Arrow on cuvette must be facing you LDH Activity (pg 14 Lab Manual) ⬜ [ΔA/min] = (initial absorbance – final absorbance) / 3 ◼ ⬜ [ΔA/min] is the change in absorbance per minute during the first three minutes of the reaction LDH Activity (μmol/min/mg) = ([ΔA/min] / 6.22 x 78,780 6.22 is the molar extraction coefficient for NADH (i.e., how strongly NADH absorbs light at 340nm) ◼ 78,780 is the dilution factor for the assay (i.e., the final ratio of muscle tissue to liquid) ◼ Upcoming Metabolism Lab ⬜ Volunteers needed (one male + one female)