Lab training Enzyme Kinetics & Photometry

Transcription

1 Lab training Enzyme Kinetics & Photometry Qing Cheng Biochemistry Division, MBB, KI Lab lecture Introduction on enzyme and kinetics Order of a reaction, first order kinetics Michaelis-Menten kinetics K M, V max and k cat Lineweaver-Burk plot Enzyme inhibition, competitive and non-competitive inhibition Spectrophotometer and Beer-Lambert Law Lab procedure Lab execution Lab report Safety in the lab 1

2 Introduction Enzyme Enzymes are biological catalysts characterized by Catalytic efficiency Specificity Enzyme Substrate Regulated activity Enzyme-catalyzed reactions are affected by Enzyme concentration Substrate concentration Temperature ph Inhibitors Activators Free energy Transition state, S Product ΔG (without enzyme) ΔG (with enzyme) Substrate ΔG for the reaction Product Reaction progress Introduction Kinetics Kinetics is the study of chemical reaction rate (v, stands for velocity) Enzyme kinetics is the study of enzyme catalyzed reaction Determination of kinetics parameter measurement of enzyme activity First order kinetics vk S Zero order kinetics vk S 2

3 Introduction Order of reactoin First order reaction Reaction rate is proportional to the substrate s concentration. This is true when the substrate concentration is low during the reaction, and the substrate is the determine factor for the reaction rate. Zero order reaction The reaction rate is independent of the substrate concentration. This is true when the substrate concentration is much higher than the enzyme concentration during the reaction, and the enzyme is the determine factor for the reaction rate. S k P Reaction rate, v Zero order reaction k First order reaction Substrate concentration [S] Michaelis-Menten kinetics To understand how enzyme functions, we need a kinetic description of their activity. The reaction rate rises linearly as substrate concentration increases (first order reaction) and then begins to level off and approach a maximum at higher substrate concentration (zero order reaction) For many enzymes, the reaction rate V 0 is defined as the number of moles of product formed per unit time when [P] is low, that is at times close to zero (hence, V 0 ) V 0 [S 4 ] Reaction rate, v Zero order reaction Reaction rate, v V 0 [S 3 ] V 0 [S 2 ] V 0 [S 1 ] First order reaction Substrate concentration [S] Time 3

4 Michaelis-Menten kinetics Consider an enzyme that catalyzes the S to P by the following pathway: V 0 is measured when [P] is low, therefore k 4 becomes negligible. V 0 = k 2 [ES] Rate of ES formation =k 1 [E][S] Rate of ES breakdown = (k 2 +k 3 )[ES] Steady state: When [ES] is formed and broken down at the same speed Michaelis-Menten kinetics Simplify the previous equation by define a new constant, K M, called Michaelis constant Solving [ES] K M has the units of concentration K M is independent of either [E] or [S] (because at maximum rate, [ES]=[E tot ]) 4

5 Michaelis-Menten kinetics Reaction rate The lower the K M value The more efficient the enzyme Substrate concentration [S] At very low substrate concentration ( ): The reaction rate is directly proportional to the substrate concentration At very high substrate concentration ( ): The reaction rate is maximal, independent of substrate concentration. When K M is equal to the substrate concentration ( ): K M is equal to the substrate concentration at which the reaction rate is half its maxim value Lineweaver-Burk plot V max is difficult to estimate because the initial reaction rate approaching V max asymptotically with increasing substrate concentration. In addition, the high concentration of substrate often inhibits reaction rate. To solve this problem, Lineweaver and Burk (1934) had inverted the Michaelis- Menten equation, which is referred as Lineweaver-Burk plot (or Double reciprocal plot): y = ax + b In this equation, 1/V and 1/[S] are variables, while K M /V max and 1/V max are constants. This can be plotted as a linear equation (y = ax + b). Specifically, 1/v is y, 1/[S] is x, K M /V max is a (slope) and 1/V max is b (yintercept). We can accurately calculate K M and V max value from a Lineweaver- Burk plot. 5

6 Nonlinear regression Indeed, K M and V max values can be calculated directly from the Michaelis-Menten equation through nonlinear regression. y = ax/(b+x) (in short: Input the data pairs (V and [S]) Choose fit function: ax/(b+x) Leave Parameter guesses as it is. Choose Convergence Mode: Damped or Strongly damped Click Fit or Fit & Plot (Java needed for plotting) Nonlinear regression Indeed, K M and V max values can be calculated directly from the Michaelis-Menten equation through nonlinear regression. y = ax/(b+x) (in short: Input the data pairs (V and [S]) Choose fit function: ax/(b+x) Leave Parameter guesses as it is. Choose Convergence Mode: Damped or Strongly damped Click Fit or Fit & Plot (Java needed for plotting) 6

7 Inhibition of enzyme activity Competitive inhibition Noncompetitive inhibition Substrate Competitive inhibitor Noncompetitive inhibitor Enzyme Enzyme Enzyme Inhibition of enzyme activity Competitive inhibition I S E + S ES E + P + I k i Competitive inhibitor Enzyme EI No inhibitor Competitive inhibitor + Competitive inhibitor No inhibitor V max is not affected K M is increased 7

8 Inhibition of enzyme activity Noncompetitive inhibition S I E + I ES E + P k i S EI EIS Noncompetitive inhibitor Enzyme No inhibitor Noncompetitive inhibitor No inhibitor + Noncompetitive inhibitor V max is decreased K M is not affected Inhibition of enzyme activity Competitive inhibitor Noncompetitive inhibitor Mix inhibition No inhibitor No inhibitor No inhibitor V max is not affected K M is increased V max is decreased K M is not affected V max is decreased K M is increased 8

9 Photometry - Spectrophotometer How to measure the chemical reactions rate Different molecules have different absorption Some molecules (e.g. proteins) have several absorbance peaks during the wave scan Spectrophotometer Light source Filter Sample Detector Readout Photometry - Beer-Lambert law Beer-Lambert law is a mathematical means of expressing how light is absorbed by matter. The law states that the amount of light emerging from a sample is diminished by three physical phenomena: The concentration of absorbing sample in its pathway (C, in unit of molarity, M) The distance the light travels through the sample (l, in units of centimeters, cm) The probability that the light of that particular wavelength will be absorbed by the material, also known as molar absorption (or extinction) coefficient (ε), in units that are reciprocals of molarity and distance in centimeters, M -1 cm -1 ) T: 0-1 A: - 0 Due to technical limitation, the best reading range of spectrophotometer is normally from 0.1 1, thus: o o If A is too high, dilute the sample If A is too low, concentrate the sample 9

10 Lab training Enzyme: Alkaline Phosphatase (ALP) Remove phosphate groups from many types of molecules. Function as a dimer, and take effect under alkaline conditions Made in liver, bone, and other tissues. It can be measured in a routine blood test. Abnormally high serum levels of ALP may indicate bone, liver disease, etc. Lab training Outline Determine the following parameters of alkaline phosphatase using p-nitro-phenyl-phosphate (NPP) as substrate Optimal ph K M V max Inhibition p-nitro-phenyl-phosphate (NPP) p-nitrophenol 10

11 Lab training Maximal absorbance Determine the maximal absorbance (λ max ) for p-nitrophenol Set wavelength scan in the range of nm Calculating molar absorption coefficient (ε) using Beer-Lambert law Lab training Incubation time The effect of incubation time on the amount of product formed Incubation at 40⁰C water bath Incubation time (9 different time from min) Measure absorbance within 15 min at the end of the reaction Plot: amount of product formed (y-axis) against time (x-axis) dh 2 O NFF Buffer with ph 9.5 Testing tubes ( 9) Add enzyme Blank tubes ( 9) Add dh 2 O 11

12 Lab training Optimal ph The effect of ph on the reaction rate (v) Incubation at 40⁰C water bath Incubation time: 10 min Measure absorbance within 15 min at the end of the reaction Plot: amount of product formed (y-axis) against time (x-axis) dh 2 O NFF Buffer with differnt ph Testing tubes ( 9) Add enzyme Blank tubes ( 9) Add dh 2 O Lab training K M, V max, Inhibition Determine the K M and V max value for alkaline phosphatase, and the inhibition type of the inhibitor provided Incubation at 40⁰C water bath Incubation time: 10 min Measure absorbance within 15 min at the end of the reaction Michaelis-Menten plot: Reaction rate (y-axis) against [S] (a-axis) Lineweaver-Burk plot: 1/V (y-axis) against 1/[S] (a-axis) Determine K M,V max, and inhibition type Each series contains 6 tubes with different substrate concentration [S] a b c d a. With enzyme only b. With enzyme and 1 st inhibitor (0.5 mm phosphate solution) c. With enzyme and 2 nd inhibitor (1 mm phosphate solution) d. Blank (with neither enzyme nor the inhibitor) 12

13 Lab training Case study (ADH) K M determination for alcohol dehydrogenase Determine K M, V max, of alcohol dehydrogenase using either methanol or ethanol as substrate from the values in the lab compendium. Determine inhibition type of ethanol on ADH when using methanol as substrate. Calculate the blood alcohol content (BAC) in the patient undergone ethanol treatment. Lab note and reports Write lab notes carefully, which should be approved and countersigned by your teaching assistant. A written lab report is needed Brief introduction and aim Brief description of the execution of the lab work Results including all the raw data, calculations including the equation, and all the plots. Don t forget to specify units in calculations and plots Brief conclusions and discussion Answer all the questions in the compendium. 13

14 Lab safty Lab coat, goggles, gloves No eating or drinking in the lab Mouth pipetting is prohibited Prevent accidents Be well prepared Work calmly When you leave the lab Wash hands Remove lab coat Use your judgment Ask teaching assistant Question? 14