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 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

• K2 also known as kcat

• At steady state– [ES] = (k1/k-1 + k2) [E] [S]

k-1k2

k1

km: A ratio of Rate constantspage 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+ CH3CH2O + H+

+ NADHWe 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]

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), V0

Lineweaver-Burk (Figure C.1-6) page 86-88– Determine basic properties of enzyme KM

• Part two Page 89-92– Effects of concentration, temperature and pH

• Report requirements: Page 102-104.

Experiment 1: Page 86&76

1.0

2.0

3.0

060 120 180 240 300 360 4200

time (seconds)

y = 0.0191 x - 1.0067

Kinetic curve.

Add enzyme

Experiment 2 Page 87-88Determine 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 (ml)

Buffer Ethanol [S] NAD+ ADH V 1/V 1/[S]

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

Data table Page 87Km

Initial Velocity (Vo page 85)

1 23

45

time (sec)

[P] (M)

15 45

V0 (M/sec)

[E] (M)

Observed trend

Trend ofactual tangental slopes of progress curves with increased [E]

A

B

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 25 points

Clean up and Check outPage 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

Kinetics Write Up

• See report outline Page 102

• Remember describe what happened in your experiment

Next time

Examine the effects of:Temperature

pHSubstrate concentration

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

V0,max(T)

T (oC)4020 30 50 60 70 80

Arrhenius kinetic behavior

proteindenaturation

Effect of pH page 99

V0

pH

pKa of reaction 1 ~ 4.0

pKa of reaction 2 ~ 9.0

2 124 6 8 10

max

low

Activity decreases due to lysine deprotonation

Activity decreases due to glutamate/aspartate protonation

Maximal activity range

Extra Credit for this Lab5 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

V0,max(T)

T (oC)4020 30 50 60 70 80

Arrhenius kinetic behavior

proteindenaturation

Arrhenius plot

Slope = _Ea/R

R= 1.9872041(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

• http://en.wikipedia.org/wiki/Dehydrogenase• What generalizations can you make regarding

your observations on ADH and the properties of these other enzymes.