Steele - Lecture 4

Question 0

Why is the displacement of water in active sites by substrates important?

Answer 0

Because they make hydrophobic interactions possible

Question 1

In a Lineweaver-Burke Plot, what is the x-intercept, y-intercept, x-axis, y-axis, slope?

Answer 1

x-axis: 1/S
y-axis: 1/Vo
x-intercept: -1/Km
y-intercept: 1/Vmax
Slope: Km/Vmax

Question 2

What kinds of residues are in an active site?

Answer 2

Binding residues (help substrate situate in AS)

Catalytic residues (perform catalytic function, stabilizing transition state between Substrate and Product)

Question 3

Why are multiple contacts in an AS important?

Answer 3

To ensure proper orientation of substrate in AS

Question 4

What is the difference between induced-fit and lock-key models?

Answer 4

Induced fit includes a conformational change in the enzyme induced by substrate binding. This conformational change helps stabilize the ES complex.

Question 5

True or false: Catalytic residues are often found in single domain in a protein with 4D structure?

Answer 5

False- In a protein with 4D structure, AS generally shared between 2 subunits

Question 6

True or false: Catalytic residues are often nucleophiles

Answer 6

True.

Question 7

Why do catalytic sites often have residues with atypical pKa?

Answer 7

Because their immediate environment in the protein results in an atypical pKa

Question 8

True or false: active site amino acids are found in tandem

Answer 8

False, amino acids in AS are sometimes found distant in primary sequence. Form AS through 3D structure

Question 9

What residues are involved in LDH AS?

Answer 9

1) His- proton donor, its imidazole group is involved in acid-base catalysis
2) Asp- Sabilizes His
3) arg109- helps polarize the carbonyl group through a + charge on arg; helps stabilize transition state
4) Arg171- binding residue thru electrostatic interactions

Question 10

Describe in general terms the AS mechanism of chymotrypsin (a serine protease)

Answer 10

The serine OH is partially deprotonated, making the O a nucleophile. This leads to the release of 1/2 the polypeptide and the retention of the other half thru a covalent bond to serine (tetrahedral intermediate). The covalent bond is severed through a hydrolysis reaction, leading to release of the other half of the polypeptide (in the process another tetrahedral intermediate is formed). Inevitably, the OH on serine is restored.

Question 11

What are substrates that can bind the AS of a protein?

Answer 11

Analogs

Question 12

How can an enzyme achieve loose or tight specificity?

Answer 12

By the residues located in the AS pocket. Ex: carboxylate from aspartic acid will attract + charged residues; ex: hydrophobic R groups from valine will attract hydrophobic substrates, and can occlude the pocket to allow only small chains in

Question 13

Are enzyme inhibitors reversible or irreversible?

Answer 13

Can be both

Question 14

What are two categories of reversible inhibtion?

Answer 14

1) competitive inhibition: competes directly with binding of S to active site
2) non-competitive: acts on allosteric site different than AS; indifferent to presence or absence of S

Question 15

How does competitive inhibition affect Km or Vmax? what is an example?

Answer 15

Vmax unchanged because substrate can dilute out inhibitor as substrate concentration increases

Km increases

ex: prevastatin binding of HMG CoA reductase

Question 16

How does noncompetitive inhibition affect Vmax and Km?

Answer 16

Km unaffected, Vmax decreases because inhibitor is indifferent to substrate

Question 17

How can irreversible inhibition be overcome?

Answer 17

It can’t really, even with high S (because of covalent modification of enzyme)

Can only be overcome by new protein synthesis

Question 18

What are 2 examples of reversible inhibitors?

Answer 18

1) penicillin- covalently binds transpeptidases, preventing synthesis of bacterial cell walls
2) aspirin- modifies active site serine (by acetylation) in COX-1 and COX-2, preventing inflammation

Question 19

What is a physiological of allosteric regulation involving nucleotides?

Answer 19

ribonucleotide reductase- reduces ribonucleotide diphosphates to deoxyribonucleotide diphosphates.

2 allosteric sites -
one regulates activity and one regulates specificity. as you might expect, high ATP will increase activity, high dATP reduces

one regulates substrate specificity, balancing the production of all four dNTPs

Question 20

What do you need for enzyme catalysis to take place?

Answer 20

1) correctly folded enzyme
2) co/post translational modification
3) cofactors (generally act like substrates)
4) prosthetic groups (covalently bound)
5) metal ions

Question 21

True or false: metal ions are not involved in catalysis

Answer 21

False. They can or can’t be. Generally, if metal ion is involved in catalysis, it cannot be removed if enzyme is to function

Question 22

How is collagen structured?

Answer 22

3 collagen alpha chains coiled around one another to form a helix

Question 23

What is the common primary sequence repeat found in collagen?

Answer 23

-Gly-X-Y-

X= often proline
Y= often Hydrxylysine [formed with vitamin C (ascorbate) as a cofactor]

Question 24

What causes scurvy?

Answer 24

Deficiency of Vit C intake

Results in lack of hydroxylysine, impaired collagen formation with fibers that have less tensile strength

Question 25

Describe collagen synthesis briefly

Answer 25

hydroxylysines are synthesized, glycosylated starting from the C-terminus end

collagen fibers get crosslinked to form more macromolecular structures

Question 26

What causes osteogenesis imperfecta?

Answer 26

Replacement of glycine residue by a bulky AA.

If mutation is on C-terminus of protein, disease is more lethal because gylcosylation can’t happen, preventing collagen fibers from even beginning to wrap.