Enzyme Kinetics Lab C1 Two periods Pages

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 CO 2 and H 2 O –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 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] E + S ES E + P K 2 also known as k cat At steady state –[ES] = (k 1 /k -1 + k 2 ) [E] [S] k -1 k2k2 k1k1

k m : A ratio of Rate constants page 80-81(Info Box 5) [ES] = (k 1 /k -1 + k 2 ) [E] [S] k m = k -1 + k 2 / k 1 –K m =Michaelis constant

Initial velocity V o 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, V o as [S] increases –Until rate becomes constant: approaches V max

Effect of [Substrate]

Effect of [substrate] on R X Velocity

Michaelis-Menten Equation V 0 = V max [S] K m +[S]

Lineweaver-Burk Plot Units of Km are concentration

Can calculate K m One of the most important descriptive terms in all of biology

Alcohol Dehydrogenase: ADH CH 3 CH 2 OH + NAD + CH 3 CH 2 O + 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

NADH

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 CH 3 CH 2 OH + NAD + CH 3 CH 2 O + H + + NADH 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 K m

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 V o with increasing [S] Re-plot these data in the double-reciprocal Lineweaver-Burk plot Remember Vo= Δ NADH/Δ Time.

This Lab and Next Lab Part one Kinetic Curve (Figure C.1-5), V 0 Lineweaver-Burk (Figure C.1-6) page –Determine basic properties of enzyme K M Part two Page –Effects of concentration, temperature and pH Report requirements: Page

Experiment 1: Page 86&76 Kinetic curve. Add enzyme

Experiment 2 Page 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

Table C.1-1. ____ Assay #Water ( m l) BufferEthanol[S]NAD + ADHV1/V1/[S] Data table Page 87 Km

Initial Velocity (Vo page 85)

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 Remember you are determining the optimal conditions for this assay

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 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

Kinetics Write Up See report outline Page 102 Remember describe what happened in your experiment

Next time Examine the effects of: Temperature pH Substrate concentration

Next Exercise Effects of Temp, pH and Enzyme concentration. Page Read carefully “Factors that affect catalysis” (Page )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

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 (18)×10 −3 Kcal/mol –Shows how to calculate using Xcel

Arrhenius plot Slope = _Ea/R R= (18)×10 −3 Kcal/mol

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 What generalizations can you make regarding your observations on ADH and the properties of these other enzymes.