2 Rapid Reactions: determination of Km and Vmax for YADH

why are we investigating YADH

to work out:

- initial rate of reaction

- Vmax

- Km

via UV/visible spec & MIchealis-Menten kinetics

What enzyme reaction are we studying

ethanol ⇌ acetaldehyde

catalysed by alcohol dehydrogenase

Why is YADH important

- in yeast, YADH helps with ethanol formation during fermentation - important in brewing

- in mammals, alcohol metabolism is carried out by the liver, ethanol is converted to acetaldehyde by ADH, the converted to acetate by ALDH, so acetate can enter metabolism as acetly CoA

what is this reaction pushed towards and why

ethanol + NAD+ → acetaldehyde + NADH + H+

because NADH is easy to measure at A340

- so, can follow reaction by measuring A340 over time

- NADH+ doesn't absorb strongly at 340nm, NADH must be used instead

what buffer does this reaction use and why

150mM sodium pyrophosphate buffer at pH 8.5

- a high pH buffer helps reaction proceed towards acetaldehyde + NADH

- forwards reaction makes H+, a high pH has low H+

- as reaction produces H+, alkaline buffer absorbs/ resists the H+

- because H+ is kept low, equillibrium shifts to make more products

what is the initial rate and the conditions

where the

- substrate still in excess

- very little product formed

- reverse reaction negliginle

- graph is approx linear

what is initial rate written as

V0, you calculate it from the slope of the initial linear part of the absorbance/time graph

here, we measure the first 20 seconds of the reaction

reagents

- YADH - enzyme

- 1M ethanol - substrate

- 10mM NAD+ - cofactor converted to NADH

- 150mM sodium pyrophosphate buffer pH 8.5 - maintains alcohol pH

- distilled water

independent variable

the ethanol concentration, enzyme stayy constant because you only want to test the effect of the substrate concentration

conditions of the reagents in the experiment

- ethanol volume changes

- water volume changes to compensate

- enzyme volume stays constant

- NAD+ stays constant

- buffer constant

- total cuvette colume constant

first cuvette mixture, component and volumes

what do you have to add last

the enzyme, as it starts the reaction

0.1ml YADH

what does the blank cuvette contain

before adding the enzyme, the blank cuvette contains:

- buffer

- ethanol

- water

- NAD+

everything except the enzyme, so the machine removes everything except the NADH formation

what are the spec settings

After reading, the machine gives ΔABS/Δt

(change in absorbance over time)

how is the experiment repeated

Repeated using different ethanol volumes:

- 200, 150, 100, 50, 25, 10 and 5µl

- they produce different substrate concentration in the cuvette

why is the water volume adjusted

total cuvette volume must stay the same.

if you use less ethanol, you add more water

- this helps keep the ethanol and water volume constant - a control

what is M

number of moles dissolved in exactly a litre of solute

concentration ethanol stock used

1M

what's DF

DF = total volume in cuvette / volume of ethanol added

whats the total cuvette volume

2500µl

perform an example using 200µl ethanol

how the absorbance data becomes reaction rate

1. spec gives change in absorbance over the initial reaction time

- we record change in A340 over 20s

2. need to convert to per minute.

multiple absorbance change by 3 to get ΔA/Δt per minute

Beer-Lambert

need to convert absorbance into concentration

Beer-Lambert example

what to do after calculating Beer-Lambert

repeat theh assay fro each ethanol concentration:

for each calculate

1. ethanol conc [S]

2. change in absorbance

3. rate of absorbance change, ΔA/Vt

4. initial rate, V0

then plot V0 against ethanol

what type of graph do you get when you plot V0 against ethanol

rectangular hyperbola

what is Vmax

the maximum rate of reaction

what happens to rate at a low substrate concentration

- substrate limiting

- many enzyme active sites are empty

- increasing ethanol increases reaction rate lots

- plateau Vmax

what happens to rate at a high substrate concentration

- most active sites are occupied

- enzyme becomes saturated

- increasing ethanol has less effect

- plateau Vmax

what happens at Vmax

- all active sites are occupied

- enzyme is working as fast as possible

- adding more substrate doesn't significantly increase the rate

Km

substrate concentration when reaction rate is half Vmax

so,

when V0 = 1/2 Vmax, [S] = Km

Michaelis-Menten equation

how to perform Michaelis-Menten

from the graph of V0 against [S]

1. look for the plateau

2. plateau gives Vmax

3. find substrate conc at 1/2 Vmax = Km

Lineweaver-Burk plot

transformed versionof Michaelis-Menten

- instead of plotting V0 against [S] you plot 1/V0 against 1/[S]

- gives a straight line

Lineweaver-Burk straight line equation

advantages and disadvantages of te Line-weaver-Burl plot

advantages:

- makes data linear

- can make Vmax and Km easier to estimate

disadvantages:

- can exaggerate errors, especially at low substrate concentrations

Hanes-Woolf Plot

- another linear transformation (gives a straight line)

- you plot:

S/V0 against [S)

straight line equation for Hanes-Woolf plot

difference between Hanes-Woolf & Lineweaver-Burk plot

- both are ways to turn the curved Michaelis-Menten into a straight line

- Hanes-Woolf is usually more reliable because it doesn't exaggerate low-substrate errors as badly. because it doesn't use recpricols

experiment sources of error

- inaccurate pipetting

- adding wrong water/ ethanol volume

- not adding enzyme last/ fast enough

- not mixing after enzyme added

- cuvette bubbles

fingerprints or liquid droplets on cuvette

- not rinsing cuvette between uses

- enzyme warm

- wrong blank or wavelength

whole practical summary

1. prepare reaction mixture without enzyme

2. blank spec using machine without YADH

3. add YADH last to start the reaction

4. measure A340 for 20s

5. use the initial change in A340 to calcuate ΔA/Δt

6. use Beer-Lambert law to convert ΔA/Δt into V0

7. repeat with different ethanol concentrations

8. plot V0 aganst ethanol

9. use the graph to estimate Vmax and Km

10. use lineweaver-burk or hanes-oolf to estimate Vmax & Km more clearly