exercise 4: enzyme activity and environmental effects enzyme activity and...exercise 4: enzyme...

Exercise 4: Enzyme Activity and

Environmental Effects

Learning Objectives

• Explain the function of a spectrophotometer

• Use a spectrophotometer to measure enzyme activity

• Determine the relationship between substrate concentration and the rate of the enzymatic reaction

• Determine the relationship between other environmental variables (temperature) and the rate of the enzymatic reaction

• Design and conduct an experiment to explore the effect of another variable on the rate of the enzymatic reaction

Enzyme Review

Enzymes are proteins that catalyze (facilitate) reactions.

Catalase and α-Amylase

CatalaseHydrogen

peroxide

H2O (water)

and

O2 (oxygen)

A demonstration

Carrot and Hydrogen Peroxide

• Substrates or reactants bind to the enzyme’s active site.

Hydrogen peroxideWater and Oxygen

Catalase (in carrot)

Why no bubbles before the carrot?

What does catalase NOT change?

What DOES catalase change?

Enzymes bind reactants

and stress reactants to

catalyze reactions.

This reduces the

activation energy.

What does this graph tell us about pepsin and trypsin?

Figure 8.18

Ra

te o

f re

ac

tio

n

(b) Optimal pH for two enzymes

Optimal pH for pepsin

(stomach enzyme)Optimal pH

for trypsin(intestinalenzyme)

10 2 3 4 5 6 7 8 9

What does this graph tell us about human and thermophilic

bacteria?

• Some enzymes require cofactors to function optimally. These are

either metal ions or small organic molecules called coenzymes.

• So enzymes are regulated by molecules that are not part of the

enzyme itself.

Calcium

α -Amylase

Metal ion cofactors: Na+, K+, Mg++,

Zn++, Cu++, Fe++, Fe+++

CHELATORS: bind metal ions

What affect would a chelator

have on amylase?

Substrate Concentration [S]

• The rate of product formation during an enzyme-

catalyzed reaction increases linearly at low [S].

• The reaction rate reaches maximum speed at high [S]

called Vmax.

Vmax = active sites on enzyme are all occupied!

Vmax

Exercise Four- Enzyme Activity

Monitor the activity of Tyrosinase (an enzyme)

1. Substrate concentration

2. Temperature

And then either:

Enzyme concentration

Chelator (EDTA) or

Sodium chloride or

Tyrosinase is an enzyme that catalyzes

the biosynthesis of the pigment

melanin from the amino acid tyrosine.

• Skin coloration

• Browning of fruits

Copper ionsOxygen

In the presence of oxygen (O2), tyrosinase (E) catalyzes the

hydroxylation of tyrosine into 3,4-dihydroxyphenylalanine

(DOPA).

Tyrosinase then catalyzes DOPA into dopaquinone (which

spontaneously converts into dopachrome).

You can monitor the activity of tyrosinase

by looking at the color of the solution.

Over time, what do you think will happen to the color of a

DOPA solution after the addition of tyrosinase?

a. Solution begins clear and then turns brown

b. Solution begins brown and turns clear

Dopachrome is a orange/brown

colored compound…..

As the reaction proceeds, more substrate is converted to product

(dopachrome) by tyrosinase and the solution will become darker.

Time

How do you measure the change in color??

Spectrophotometer

Cuvettes (containing the solution) are inserted into the

spectrophotometer.

•You figure out which wavelength of light is absorbed by your

product

•Select wavelength and shine it through sample.

•Start your reaction

•Measure how absorbance changes over time

You’ll be given the following to explore tyrosinase activity:

1) DOPA

2) A solution containing potato

3) SpectroVis

Where do you find the enzyme?

Where do you find the substrate?

How will absorbance values change over time?

4.1 and 4.2: Turn on Spec and Calibrate

Turn LoggerPro on.

Page 64: Prepare a blank to calibrate or ‘tare’ the spectrophotometer:

Blank has everything EXCEPT reactant (no DOPA).

Buffer

Enzyme

dH2O (in place of DOPA)

QUESTIONS:

1) Why do you need to blank the spectrophotometer?

2) Why dH2O?

4.3 Absorbance Spectrum

You have to figure out which wavelength to use to monitor

the reaction.

So you need the absorbance spectrum for dopachrome (the

product).

What is on the X axis? Y axis?

Explain why MAX around 470

nm and not 700 nm.

Select λ MAX and use it to

monitor change in absorbance

over time.

4.4 Tyrosinase activity monitored over

time

What is on the X axis? What is on the Y axis?

How is this different from the absorbance spectrum?

Why is absorbance

increasing over time?

4.5 Exploring Substrate Concentration

You will complete a serial dilution to prepare samples from a

stock solution of DOPA.

Exploring Substrate Concentration

Prepare cuvettes with a range of [DOPA].

After adding enzyme, you will measure change in color over

time (Absorbance vs. Time) and determine the rate of the

reaction for EACH concentration.

How will the rate of the reaction change with increasing

substrate concentration?

Why?

You will design and conduct an experiment to

determine the effect of the following on

tyrosinase:

Enzyme concentration

EDTA (chelator)

Sodium chloride

What do you expect the effect increasing enzyme

concentration will be on absorbance values? EDTA?

Sodium chloride?

How does enzyme concentration affect the reaction?