Chymosin Lab

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.

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)

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

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

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

Enzymes make it easier for reactions to happen

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

Lowering the activation energy makes enzyme-catalyzed reactions faster

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

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

Enzyme Inhibition- Enzymes can be sped up or slowed down

Protease inhibitors make enticing HIV drug candidates

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

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

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.

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

b - galactosidase galactose lactose b - galactosidase glucose H O 2 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