Biology 1107 Enzymes

What was the molarity of the stock solution of p-nitrophenol?

0.25 mM or 0.25 µmoles/mL (micromoles)

How come non-compatible substrates are not able to bind with the enzyme's active site?

Because they don't have the right shape. See animation: http://phschool.com/science/biology_place/labbench/lab2/binding.html

Which factor, pH or temperature, affects OPTIMUM performance of an enzyme? Give two examples at opposite ranges.

Each enzyme functions best within a certain pH range. For example, the enzyme pepsin, which works in your stomach, functions best in a strongly acidic environment. Lipase, an enzyme found in your small intestine, works best in a basic environment.

What do enzymes do?

Enzymes catalyze reactions by lowering the activation energy necessary for a reaction to occur.

What kind of proteins are enzymes?

Globular proteins

How does irreversible inhibition differ from irreversible enzyme inactivation?

Irreversible inhibitors are generally specific for one class of enzyme and do not inactivate all proteins; they do not function by destroying protein structure but by specifically altering the active site of their target. For example, extremes of pH or temperature usually cause denaturation of all protein structure, but this is a non-specific effect. Similarly, some non-specific chemical treatments destroy protein structure: for example, heating in concentrated hydrochloric acid will hydrolyse the peptide bonds holding proteins together, releasing free amino acids.

What happens to the speed of reactions relative to temperature?

Reactions happen faster with higher temperatures, but only up to the optimal temperature. Too hot of a temperature will denature an enzyme.

How is the active site created on an enzyme?

The active site of an enzyme is created by their folded conformation.

Explain the concept of induced fit.

The change in shape of the active site of an enzyme so that it binds more snugly to the substrate, induced by entry of the substrate. Induced fit enhances catalysis, as the enzyme converts substrate to product.

What will happen if you forget to add NaOH to the tube?

The enzyme reaction will continue and the solution will remain colorless.

What happens as pH moves away from it's optimal range?

When the pH changes, the active site progressively distorts and affects enzyme function

Describe Non-competitive inhibition

a form of mixed inhibition where the binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate. As a result, the extent of inhibition depends only on the concentration of the inhibitor. Vmax will decrease due to the inability for the reaction to proceed as efficiently, but Km will remain the same as the actual binding of the substrate, by definition, will still function properly.

How can ACP be detected? What alternative substrate is used?

by a colorimetric assay using p-nitrophenyl phosphate

how do irreversible inhibitors work?

covalently modify an enzyme, and inhibition can therefore not be reversed

What do you call an enzyme that lost it's conformational shape?

denatured

what product is produced by a reaction between ACP and p-nitrophenyl phosphate?

p-nitrophenol

Write the reaction equation for this reaction and describe what happens and what measurement is quantified at the end.

p-nitrophenyl + H₂O →ACP→ p-nitrophenol + P ↓ add NaOH to stop reaction, p-nitrophenol is yellow, quantify by spectrophotometry

What two factors can affect the conformation of an enzyme?

pH and temperature

Competitive inhibitors are often _________

similar in structure to the real substrate

Describe competitive inhibition

substrate and inhibitor cannot bind to the enzyme at the same time the inhibitor having an affinity for the active site of an enzyme where the substrate also binds; the substrate and inhibitor compete for access to the enzyme's active site

What can happen when an enzyme's conformation is significantly altered because of pH or temperature variation?

the enzyme may no longer catalyze reactions it becomes denatured

Each enzyme is specific for __________

the reaction it will catalyze.

What is the molecule that the enzymes act on called?

the substrate

What maintains the conformation of an enzyme?

the various amino acids that compose it,

What is the source of the enzyme acid phosphatase (ACP)?

wheat germ

4 steps of an enzyme catalyzed reaction

1 Substrate and enzyme are available 2. Substrate binds to enzyme's active site 3. Substrate is converted to products 4. Products are released, enzyme is unchanged and may catalyze another reaction cycle

What two things can be correlated with the amount of product formed which is found from the spectrophotometer reading?

1. Amount of enzyme present 2. Level of activity of the enzyme

Name two functions of acid phosphatase?

1. Cleave the phosphate group. 2. Act in an acid environment.

Four types of reversable inhibitors

1. Competitive 2. Uncompetitive 3. Mixed 4. Non-competitive

How does adding NaOH cause inhibition?

don't know

What are some reasonable sources of error in this experiment?

don't know

Four factors that affect the activity of an enzyme

1. Enzyme concentration 2. Substrate concentration 3. pH 4. Temperature

What 5 pathological conditions in animals and people can arise from too much acid phosphatase?

1. Prostate cancer 2. Myocardial infarction (heart attack) 3. Liver disease 4. Sickle-cell anemia 5. Paget's disease

Look at the burette. What two readings do you get from it. How much liquid was lost? http://i576.photobucket.com/albums/ss202/robwinfield35/Biology_1107/burette_unlabeled.gif

3.30 mL and 3.90 mL and 0.6 mL was used

What wavelength is the spectrophotometer set to?

410 nm

How long was the reaction allowed to proceed? at what temperature?

5 minutes at room temperature.

What is the rule of thumb for reading a burette or graduated test tube?

from the bottom of the meniscus

What happens in an enzyme mediated reaction?

substrate molecules are changed, and product is formed.

How can competitive inhibition be overcome?

sufficiently high concentrations of substrate (Vmax remains constant), i.e., by out-competing the inhibitor

what happens in allosteric inhibition?

the inhibitor binds to another site besides the active site and changes the overall conformation of the enzyme, thus inhibiting reactions by partially denaturing the enzyme

How does changing the enzyme concentration affect the amount of product (p-nitrophenol) produced? Explain?

Higher enzyme concentrations means more active sites that substrate molecules can bind to, thus more reactions, thus more p-nitrophenol product

How does temperature affect the activity of the enzyme? What temperature was optimal?

Higher temperature makes the reactions go faster until it is so hot that it denatures the enzyme. The optimal temperature was 50°C

What happens after the reaction and the products are released from the enzyme?

Release of the products restores the enzyme to its original form. The enzyme can repeat this reaction over and over, as long as substrate molecules are present.

What makes an enzyme specific for only one type of substrate?

The nature and arrangement of amino acids in the active site make it specific for only one type of substrate.

How does pH affect the activity of the enzyme ACP? What pH is optimal for this enzyme?

The optimal pH is 5 because when a pH buffer of 5 was used, it produced the most product

What are two common sources of error in this experiment?

The reaction is to proceed for a specific time and then stopped by addition of a strongly alkaline solution, NaOH. The yellow color of the product is enhanced by the alkaline solution. If you let the reaction go on for too long, or if you forget to add the NaOH at all.

Describe mixed inhibition

the inhibitor can bind to the enzyme at the same time as the enzyme's substrate. However, the binding of the inhibitor affects the binding of the substrate, and vice versa. This type of inhibition can be reduced, but not overcome by increasing concentrations of substrate. Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this allosteric site changes the conformation (i.e., tertiary structure or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced.

When you are doing the inhibition experiment, why do you only add buffer, enzyme and inhibitor, but wait 1 minute to add substrate?

to give the inhibitor time to occupy the enzyme active sites

Describe uncompetitive inhibition, also known as anti-competitive inhibition

when an enzyme inhibitor binds only to the complex formed between the enzyme and the substrate (the E-S complex). This reduction in the effective concentration to the E-S complex increases the enzyme's apparent affinity for the substrate through Le Chatelier's principle (Km is lowered) and decreases the maximum enzyme activity (Vmax), as it takes longer for the substrate or product to leave the active site. Uncompetitive inhibition works best when substrate concentration is high. An uncompetitive inhibitor need not resemble the substrate of the reaction it is inhibiting.