Transcript PPT Enzyme Kinetics (2006) – NC State Biochemistry

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]
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 concentration, 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]
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 25 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
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 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
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.