Enzyme Kinetics

Lab C1

Two periods

Pages 65-91

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– Measurement accuracy– Spectrophotometry

• Relationship between concentration and absorbance

• Will do a basic experiment in enzymology which will prepare you for a protein purification.

Enzymes

• Living organisms must be able to carry out chemical reactions which are thermodynamically very unfavorable– Break and form covalent bonds– Move large structures– Effect three dimensional structure– Regulate gene expression

• Do so through catalysis

Effect of enzymes

• A common biological reaction can take place without enzyme catalysis– Will take 750,000,000 years

• With enzyme 22 milliseconds• Even improvement of a factor of 1,000

would be good– Only 750,000 years– Living systems would be impossible

Sucrose

• A bag of sugar can be stored for years with very little conversion to CO2 and H2O

– This conversion is basic to life– When consumed is converted to chemical

energy very fast

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 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=d[B]/dt• d=change

– V=-d[A]/dt

• V=d[B]/dt = -d[A]/dt = k[A]• Large k rapid Rx• Small k slow Rx

Catalysis

• Simple reaction A [s] B [P]

• E + S ES E + P

• At steady state

• [ES] = (k1/k-1 + k2) [E] [S]• K2 also known as kcat

k-1k2

k1

km: A ratio of Rate constantspage 71-74

• [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 rate of reaction, Vo as [S] increases

– Until rate becomes constant: approaches Vmax

Effect of [Substrate]

Effect of [substrate] on RX

Velocity

Vmax = k2 [E]t

[E]t =total enzyme

=[E] +[ES]

Michaelis-Menten Equation

V0 = Vmax [S]

Km +[S]

Lineweaver-Burk Plot

1 Km 1V0 Vmax [S] Vmax

Can calculate Km

•One of the most important descriptive terms in all of biology

Lineweaver-Burk Plot

1 Km 1V0 Vmax [S] Vmax

Alcohol Dehydrogenase: ADH

CH3CH2OH + NAD+ CH3CH2O + H+

+NADH

Catalyses conversion of ethanol to aldehyde using co-enzyme NAD+

MH2 + NAD+ → NADH + H+ + M: + energy, where M is a metabolite.

NAD+ oxidized to NADH reduced

NAD+ to NADH

NAD+

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

CH3CH2OH + NAD+ CH3CH2O + 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 activityKm

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

This Lab and Next Lab

• Part one Kinetic Curve, V0 Lineweaver-Burk page 76-78– Determine basic properties of enzyme KM

• Part two Page 78-80– Effects of concentration, temperature and pH

• Lab requirements: Page 90-91.

Experiment 1: Page 73&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

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 77

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

Initial Velocity (page 75)

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

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 outPage 89

• 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 90

• 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 78

• Read carefully 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 82

V0,max(T)

T (oC)4020 30 50 60 70 80

Arrhenius kinetic behavior

proteindenaturation

Effect of Enzyme concentration page 84

I

II

[E]std

[P]

time (min)0 2 4 6 8 10

Effect of pH page 87

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

Report

• Be sure to read section 3 page 82 to 89– Has an excellent description of theory– Use in interpreting your results