Enzyme Kinetics Lab C1 Two periods Pages 73-104 Protein Chemistry • This begins a 6 day journey into the field of protein chemistry • You will learn a set of basic tools and protocols which will be important in the successful outcome. You have practiced – Measurement accuracy – Spectrophotometry • Relationship between concentration and absorbance • Today we will do a basic experiment in enzymology which will prepare you for a protein purification. Enzymes • Living organisms must be able to carry out events which are thermodynamically very unfavorable – Break and form covalent bonds – Move large structures – Effect three dimensional structure – Regulate gene expression • Do so through use of Enzymes Effect of enzymes • A bag of sugar can be stored for years with very little conversion to CO2 and H2O – This conversion is basic to life • This common biological reaction can take place without enzyme catalysis – Will take 750,000,000 years • Even improvement of a factor of 1,000 would be good – Only 750,000 years – Living systems would be impossible • With enzyme 22 milliseconds Catalysis • Carried out by very highly specialized class of proteins: Enzymes – Specialized to perform specific chemical reactions – Specialized to work in specific environments Enzymes • Have immense importance in a number of fields. – Genetic diseases are frequently defects in enzymes or increased/decreased levels of enzymes • Important diagnostic tools – Drugs exert effects by interacting with enzymes • MAO inhibitors – Used in food processing and in chemical industry – Enzyme inhibitors are a foundation of biological weapons Enzymes • A major aspect of experimental biochemistry is the purification and characterization of proteins that are enzymes – Chemical characterization – Physical characterization In the next six laboratories • You will go through the basic protocols that are used to purify and characterize catalytic proteins • The basic procedures are ones which you will use the rest of your career if you choose a career in biochemistry, molecular biology, biophysics, biochemical genetics, pharmacology, cell biology, etc…………… Kinetics • Is the science that describes the properties of a chemical reaction including those mediated by enzymes (catalysis) • Measures changes in the concentration of substrate and/or products of a reaction with time to determine the velocity of the reaction • Measures the effects of concentration, temperature, pH etc. to characterize the properties of the enzyme catalyzing the reaction Stickase From Lehninger; third edition Enzyme Kinetics • An approach to understanding the mechanism of action of enzymes • An approach to understanding how mutations may effect function • An approach to understanding how changes in the physical and chemical environments change function Rate Constant: k • A B • Velocity of Rx V=Δ[B]/Δt V=-Δ[A]/Δt • V=Δ[B]/Δt = -Δ[A]/Δt = k[A] Units are quantity/unit time e.g. Moles/Second • Large k rapid Rx • Small k slow Rx Catalysis • Simple reaction A [s] B [P] k1 • E+S ES E+P k-1 k2 • K2 also known as kcat • At steady state [ES] = (k1/k-1 + k2) [E] [S] km: A ratio of Rate constants page 80-81(Info Box 5) • [ES] = (k1/k-1 + k2) [E] [S] • km= k-1 + k2/ k1 Km =Michaelis constant Initial velocity Vo • When enzyme is mixed with high concentration of substrate [S] reaction goes rapidly to steady state. – Does not allow characterization • Use low starting [S] and increase • Hold [enzyme] constant • Measure initial rate of reaction, Vo as [S] increases – Until rate becomes constant: approaches Vmax Effect of [Substrate] Effect of [substrate] on RX Velocity Michaelis-Menten Equation V0 = Vmax [S] Km +[S] Lineweaver-Burk Plot Units of Km are concentration Can calculate Km •One of the most important descriptive terms in all of biology Alcohol Dehydrogenase: ADH CH3CH2OH + NAD+ CH3CH2O + H+ +NADH Catalyses conversion of ethanol to aldehyde using co-enzyme NAD+ NAD+ oxidized to NADH reduced NAD+ NAD+ to NADH Absorbs at λ 340 Reaction is complex • ADH +ALC ADH-ALC • ADH + NAD ADH-NAD • ADH-NAD +ALC ADH-NAD-ALC • We are not looking at this Alcohol Dehydrogenase CH3CH2OH + NAD+ + NADH CH3CH2O + H+ We will measure the forward Rx (k 2)as increased absorbance at 340. Only NADH absorbs at this wave length (page 70) Will find the assay conditions which produce max activity and calculate Km WHAT ARE WE MEASURING ? • Production of NADH NAD+ NADH Wavelength shift • Depends on participation of Alcohol and ADH • How can you do this • Ensure that NAD is not a rate limiting component. [NAD] constant and high [ADH] constant [ETOH] low and increasing Measure Vo with increasing [S] Remember Vo= Δ NADH/Δ Time. Re-plot these data in the double-reciprocal Lineweaver-Burk plot This Lab and Next Lab • Part one Kinetic Curve (Figure C.1-5), V0 Lineweaver-Burk (Figure C.1-6) page 8688 – Determine basic properties of enzyme KM • Part two Page 89-92 – Effects of temperature and pH • Report requirements: Page 102-104. Experiment 1: Page 86&76 Add enzyme Kinetic curve. Experiment 2 Page 87-88 Determine Km and Vmax • Pipetting accuracy and timing is critical • Clean cuvette – Can check clean by adding all components except ADH and placing in spectrophotometer – Absorbance should not change with time Km Data table Page 87 Table C.1-1. ____ Assay # Water Buffer Ethanol [S] NAD+ ADH ( m l) 1 0.000 0.700 2.100 0.100 0.100 2 0.600 0.700 1.500 0.100 0.100 3 1.100 0.700 1.000 0.100 0.100 4 1.600 0.700 0.500 0.100 0.100 5 1.900 0.700 0.200 0.100 0.100 6 2.000 0.700 0.100 0.100 0.100 7 2.050 0.700 0.050 0.100 0.100 8 2.080 0.700 0.020 0.100 0.100 9 2.090 0.700 0.010 0.100 0.100 10 2.095 0.700 0.005 0.100 0.100 V 1/V 1/[S] Be careful • 15 sec and 45 sec – Read same and low = • too little substrate • Didn’t add enzyme – Read same and high • Reaction is over • Contaminated one of your solutions with enzyme • Did not clean cuvette from previous assay Initial Velocity (Vo page 85) Sample data • Kinetic curve Figure C.1-5 • Lineweaver-Burke Plot Figure C.1.6 This Lab • 2 Lab periods • Pre Labs 6 points • Lab Report 20 points Clean up and Check out Page 101-102 • Return pipetters to rack • Check that you have not left cuvette in spec – Clean any spill in spec • Clean & rinse the cuvette • Clean and rinse test tubes • Throw all waste in trash Next time Examine the effects of: Temperature pH Next Exercise • Effects of Temp, pH and Enzyme concentration. Page 89-92 • Read carefully “Factors that affect catalysis” (Page 93-101)prior to coming to lab. • Lab report on Enzyme Kinetics due at start of protein purification – Remember to find the Km of another enzyme and compare it to ADH Temperature Dependence page 94 Effect of pH page 99 Maximal activity range pKa of reaction 1 ~ 4.0 pKa of reaction 2 ~ 9.0 max V0 Activity decreases due to lysine deprotonation Activity decreases due to glutamate/aspartate protonation low 2 4 6 8 pH 10 12 Extra Credit for this Lab 5 points • At lower temperatures the kinetic rate change with temperature demonstrates Arrhenius behavior • Arrhenius Plot: Plot log Vo versus 1/T degrees Kelvin, determine activation energy – Should result in a straight line – Slope = Ea (activation energy)/ R (Gas constant 1.9872041(18)×10−3 Kcal/mol • https://www.youtube.com/watch?v=Brf-_oyLFGw – Shows how to calculate using Xcel Arrhenius plot Slope = _Ea/R R= 1.9872041(18)×10−3 Kcal/mol Kinetics Write Up • See report outline Page 102 • Remember describe what happened in your experiment In your report • Emphasize what you have learned about the enzyme alcohol dehydrogenase – Its maximum velocity • Its ability to produce product at steady state – Its Km • How efficient is ADH in forming the ES complex – How does it compare to other enzymes – Its optimal pH • In what environment does it function best – Its optimal temperature – Its activation energy (if calculated) • How many Kcals or Joules are required to produce a mole of ethyl aldehyde Look at the family of Dehydrogenases • http://en.wikipedia.org/wiki/Dehydrogenase • What generalizations can you make regarding your observations on ADH and the properties of these other enzymes.