Proteins and Amino Acids

Question 1

How are ligands attracted to certain enzyme’s active sites?

Answer 1

By the size, charge, shape, hydrophilicity or hydrophobicity.

Question 2

Cooperative binding

Answer 2

When bind affects the affinity of the next binding.
The degree of cooperative binding by looking at the hill coefficient.
n > 1, positive cooperativity
n<1, negative cooperativity
n=1, enzyme doesn’t exhibit cooperativity

Question 3

What two models exist about cooperative binding?

Answer 3

MWC model - Enzymes exist in R and T states in equilibrium. When there is more ligand it shifts to more R states if more inhibitors it shifts to more T states.

Sequential model - When the ligand binds it causes a conformational in the enzyme which increases the affinity for the next ligand.

Question 4

Identify the following enzyme categories

Answer 4

• Oxidoreductases - catalyze redox reactions. ( ie. dehydrogenases)
• Transferases - transfer functional group from one compound to another ( ie. kinases- phosphorolayte)
  -Hydrolases - catalyze reaction that use water to cleave bond ( ie. kinases)
• lyases- breaks bonds w/o use of ATP or water ( ie. synthases- break bond and create a pi bond as result)
• isomerases - catalyze formation of isomers.
• ligases - combine bonds ( ie. synthetases- create bonds using ATP as energy).

Question 5

What happens to an enzymatic reaction when we add an enzyme and when we don’t?

Answer 5

With catalysts as we add more substrate it will reach a point of saturation in which more substrate doesnt change the maximum velocity. Without catalysts the rate will continue to increase.

Question 6

What two theories exist regarding ligands binding to enzymes?

Answer 6

The induced fit model - enzyme’s active site changes shape to fit the substrate ( more accepted model).

Lock and key - the enzyme’s active site and substrate fit like a “ key”

Question 7

Cofactors ( coenzymes)

Answer 7

small molecules that are attached to active site of enzyme and helps carry out it’s function.
- Apoenzyme - enzymes that doesn’t have it’s cofactor.
- Holoenzyme - enzymes that does have it’s cofactor.

Question 8

Michealis- Menten equation

Answer 8

Describes the kinetics of enzyme driven reactions.

Question 9

Describe the variables involved in enzyme kinetics.
- Km
- Kcat/Km
- Kcat ( K2)

Answer 9

• Km- michealis- menten constant. Its the substrate concentration when enzymes are operating at 1/2 Vmax. Measures affinity with an inverse relationship.
• Kcat/Km - The catalytic effeciency. Higher the value the more effecient it is.
• Kcat- turnover rate of enzyme substrate complex to enzyme and product.

Question 10

What does the lineweaver- burk plot tell us?

Answer 10

It tells us what type of inhibitor we have by looking at the changes in Vmax and Km.

Question 11

Negative feedback regulation

Answer 11

When the product of an enzyme works back on enzymes further back in the pathway and inhibit it.

Question 12

Competitive inhibitors

Answer 12

Inhibitor binds to free enzyme to the active site. Characterized by an increase in Km but with a constant Vmax. Can be outcompeted by adding more substrate.

Question 13

Noncompetitive inhibitors

Answer 13

Inhibitor binds to free enzyme and enzyme substrate complex with equal affinities Characterized by a decrease in Vmax and a constant Km.

Question 14

Mixed inhibitors

Answer 14

Inhibitors bind to free enzyme and enzyme substrate complex with different affinities. When bind to free enzyme you get an increase in Km but a decrease in Vmax. With binding to enzyme substrate complex you get a decrease in Km and Vmax.

Question 15

Uncompetitive inhibitors

Answer 15

inhibitor binds enzyme substrate complex and decreases both Km and Vmax.

Question 16

Kd

Answer 16

The dissociation constant and measures affinity. Inverse relationship to affinity.

Question 17

Irreversible inhibitors

Answer 17

An inhibitor that irreversibly binds to enzyme. Doesn’t get removed by adding more substrate only by making new enzyme.

Question 18

Allosteric sites ( allosteric activators v. allosteric inhibitors)

Answer 18

sites other than active site where inhibitor or substrate can bind,
- allosteric activator- binding causing conformational change that allows active sites to be more avaliable.
- allosteric inhibitor- binding that decreases substrate’s affinity to active site.

Question 19

Phosphorolaytion or dephosphorolaytion

Answer 19

adding phosphate group or removing phosphate group

Question 20

Glycosylation

Answer 20

Adding carbohydrate group to protein

Question 21

Zymogens

Answer 21

Enzymes are inactivated by a group in the active site until it’s removed.

Question 22

Motifs

Answer 22

proteins that arranged in a repetitive secondary structure.

Question 23

Collagen v. Elastin v. Keratins v. Actin v. Tublin

Answer 23

• Collagen - tri helical motif that plays a role in structurally supporting the EC matric.
• Elastin - responsible for stretch in EC matrix.
• Keratins - Primarily serves to mechanically support hair and nails but all epithelial cells.
• Actin - makes up myofibrils and microfilaments and has a positive and negative end by which proteins can travel unidirectionality.
• Tublin - makes up microtubules which is important for cell structure, seperation of chromosomes during mitosis, and intracellular transport via kinesin and dynein.

Question 24

Kinesins and Dyneins

Answer 24

Kinesins bring proteins down the positive end of microtubules while dyneins bring proteins toward the negative end.

Question 25

Cell adhesion molecules ( CAMs)

Answer 25

They are on the cell surface and binds the cell to the EC matrix and to other cells.
- Cadherins- glycoproteins that participate in calcium- depending binding.
- integrins - integrate into the plasma membrane and has 2 chains refered to as alpha and beta.
- selectins- bind to carbohydrate projections on other cells ( endothelial cells and WBC)

Question 26

Electrophoresis

Answer 26

A way to seperate proteins based in subjecting them to an external electric field. Smaller proteins travel further as they weigh less.
Can be native PAGE in which the protein structure is maintained or can be SDS PAGE in which the protein structure is surrounded by negative charge.

Adding a reducing agent breaks apart the disulfide bonds that make up the tertiary structure and this create multiple fragments.

Question 27

Isoelectric focusing

Answer 27

Seperating proteins based in their PI point.
Consists of a gel with a pH gradient and one side with a positive anode and a negative cathode.
Movement of the protein depends on its charge as it will move to the side with the opposite charge. They will stop when they reach their PI and they’re a zwitterion.

** Positive anode has a low pH so proteins gain protons as they approach and negative cathode has a high pH so they lose protons as they approach**

Question 28

Chromatography

Answer 28

A way to seperate proteins by their affinity to the stationary phase causing a fractionation of the samples.

• Begins by placing sample on stationary phase ( column) and then pouring the mobile phase through which will elute whatever compounds doesn’t have a high affinity to the stationary phase.

Question 29

Column Chromatography

Answer 29

A column with a polar alumina or silica beads act as the stationary phase. When we pour the mobile phase through we get bands of compounds that has different retention times.

Question 30

Ion-exchange chromatography

Answer 30

Uses a stationary phase with an ion that attracts the ion of the opposite charge.

Question 31

Size- exclusion factor

Answer 31

beads of a column that has small pores in them so that they capture small molecules while allowing bigger molecules to elute faster through therefore small molecules has a higher retention time.

Question 32

Affinity Chromatography

Answer 32

Beads are coated in something that is selective to protein of interest ( ie. receptors or antibody) everything else elutes through.

Question 33

What are the 2 ways we can experimentally find out a protein’s structure?

Answer 33

NMR spectroscopy and X- ray crystallography ( which creates a diffraction pattern by analyzing electron density around the protein).

Question 34

What are the 3 assumptions that are held when performing M-M kinetics experiments?

Answer 34

These are held true in the beginning of the reaction, thus we measure the initial rate of the reaction.

1. Free ligand assumption - substrate concentration added will be much higher than the enzyme concentration.
2. Steady state assumption - the rate of the reaction remains constant.
3. Irreversibility assumption - the products doesn’t dissociate back into reactants.

Question 35

What is salt- bridge structures in proteins?

Answer 35

A bond that’s characterized by a combination of hydrogen bonding and ionic bonding.

Important for tertiary structure.