1. Chymosin Lab

2. 1. Which variables influence enzyme performance in general. 2
1. Which variables influence enzyme performance in general? 2. With these variables in mind, hypothesize: What are the optimal conditions for the performance of chymosin? (Hint: you might investigate the normal conditions under which substances like chymosin are active). 3. Briefly describe an investigation aimed at optimizing one variable in chymosin Performance.

3. Milk contains casein Casein- German, Kase= cheese
Casein is an emulsifying protein
It is also a storage protein
Structurally similar to gluten (found in grains)

4. Cheese is made from curdled milk
Rennet is added from the 4th stomach of a calf
Rennet contains chymosin
Chymosin is an enzyme which cuts casein

5. ENZYMES They are protein catalysts
They speed up reactions inside cells
They are incredibly specific
They are incredibly efficient
-ase = “enzyme”

6. Chymosin is a protease A protease is an enzyme which cuts proteins
Specifically, it is an aspartate protease
Has an aspartate residue at its active site
HIV-Protease 1 is also an aspartate protease

7. Enzymes make it easier for reactions to happen

8. Reactions can be exergonic, yet slow- they require activation energy

9. Lowering the activation energy makes enzyme-catalyzed reactions faster

10. Enzymes can dramatically lower the energy of activation for a reaction
THE REACTION DIAGRAM
Enzymes can dramatically lower the energy of activation for a reaction
no enzyme
with enzyme E a E
Energy a
reactants
products
Reaction Course
Note that the equilibrium of the reaction is unaffected 12

11. The stages of enzyme catalysis
Substrate(s) binding
Reaction of substrate to form product(s)
Release of products
The enzyme is ready to bind the next substrate
Enzymes are unchanged by the reactions they catalyze

12. Enzyme Inhibition- Enzymes can be sped up or slowed down

13. Protease inhibitors make enticing HIV drug candidates

14. Effects of Temperature and pH
Each enzyme has an optimal temperature in which it can function
Each enzyme has an optimal pH in which it can function
Tertiary structure can be radically altered by changes in pH

15. Different enzymes work optimally at different pHs and temper-atures
Optimal temperature for
typical human enzyme
Optimal temperature for
enzyme of thermophilic
(heat-tolerant
bacteria)
Different enzymes work optimally at different pHs and temper-atures
Rate of reaction 20 40 60 80
100
Temperature (°C)
Optimal temperature for two enzymes
Optimal pH for pepsin
(stomach enzyme)
Optimal pH
for trypsin
(intestinal
enzyme)
Rate of reaction 1 2 3 4 5 6 7 8 9 10 pH
Optimal pH for two enzymes

16. 1. Which variables influence enzyme performance in general. 2
1. Which variables influence enzyme performance in general? 2. With these variables in mind, hypothesize: What are the optimal conditions for the performance of chymosin? (Hint: you might investigate the normal conditions under which substances like chymosin are active). 3. Briefly describe an investigation aimed at optimizing one variable in chymosin Performance.

17. b - galactosidase β-galactosidase is our enzyme of choice.
This enzymatic protein is a tetramer.
That means that it is comprised of how many identical subunits?
The active sites of this enzyme are located at the junctions of the subunits…here…here…here…and here.
This enzyme is produced by many different organisms and has been very thoroughly studied.
You will be studying its production in a bacteria and its activity using enzyme purified from a mold.
Transition: So what is exactly is beta-galactosidase and what does it do?… 10

18. b - galactosidase galactose lactose b - galactosidase glucose H O2
galactose
Well, Humans also produce β-galactosidase though the human version of this enzyme is called lactase.
Does anyone know what lactase deficiency is called? [No? ok./You’re right]
Let me tell you how this enzyme works:
β-galactosidase catalyzes the breakdown of a disaccharide, lactose, into two monosaccharides: glucose and galactose.
Glucose is the prime energy source for many organisms. [Any ideas now?]
In lactose intolerance, undigested lactose causes intestinal discomfort.
Transition: We know that both the amount produced and the efficiency of individual β-galactosidase are tightly regulated, which begs the question…
lactose b -
galactosidase
glucose
(aka lactase in humans) 11