Lecture 7-12

Question 1

What is the currently accepted model for enzyme substrate binding?

Answer 1

Induced fit model

Question 2

How many amino acids make up the active site and how many are directly involved in catalysis?

Answer 2

10-15 amino acids make up the active site but only 2-3 are directly involved in catalysis.

Question 3

What are the five generalizations that can be made about all active sites?

Answer 3

1. The active site is a 3D cleft or crevice.
2. The active site takes up a small volume ~10-20% of the total enzyme.
3. The active site is a unique microenvironment, normally excludes water.
4. Substrates are bound to enzymes by multiple weak interactions. Non-covalent.
5. The specificity of binding depends on the precisely defined arrangement of atoms in the active site.

Question 4

Enzymes have an equilibrium constant of that ranges from…

Answer 4

…10^2 to 10^8 M corresponding to free energy interaction ranging from -13 to -50 kJ mol^-1.

Question 5

Define the conformational selection hypothesis?

Answer 5

Suggests that enzymes exist in a variety of conformations, only some of which are capable of binding to a substrate.

Question 6

What enzyme contains no catalytic groups and is solely catalyzed by binding energy?

Answer 6

Tyrosyl tRNA synthetase

Question 7

Describe the induced fit model?

Answer 7

Enzyme binds the substrate via many relatively weak non-covalent interactions. In addition to chemical catalysis, binding energy between the enzyme and the substrate is used to stabilize the TS, lowering the activation energy, and increasing the rate of reaction.

Question 8

What is the functions of chymotrypsin?

Answer 8

Protease

Question 9

Which three residue are vital for catalytic function in chyotrypsin?

Answer 9

195-Serine, 57-Histidine, and 102-Aspartate

Question 10

Describe the two step reaction in which chymotrypsin catalyses peptide bond cleavage?

Answer 10

1. The acyl group of the substrate becomes covalently bound to the enzyme as the amide is released leaving an acyl-enzyme intermediate.
2. The acyl-enzyme intermediate is hydrolysed to release the carboxylic acid component of the substrate and regenerating the free enzyme.

Question 11

What is the role of 57-Histidine in chymotrypsin?

Answer 11

Positions the serine side chain and polarise its hydroxyl group so that it is poised for deprotonation. In the presence of the substrate His accepts the proton from the hydroxyl group of 195-serine, acting as a general base catalyst.

Question 12

What is the role of 102-Aspartate in chymotrypsin?

Answer 12

Helps better orient the his residue and make it a better proton acceptor through hydrogen bonding and electrostatic effects.

Question 13

Chymotrypsin, trypsin and elastase all catalyse specific peptide bond cleavages. What amino acids do each target?

Answer 13

Chromotrypsin = aromatic (bulky) amino acids e.g. Phe, Tyr, and Trp.
Trypsin = positively charged e.g. Arg, Lys
Elastase = small, hydrophobic e.g Ala.

Question 14

Name the three regions within the active site of serine proteases?

Answer 14

1. Activated serine site composed of the three amino acids.
2. Oxyanion hole where the unstable intermediate is stabilised by Gly and Ser backbone residues. The H on these residues contributes a positive charge that stabilises the negative charge of the intermediate.
3. Large hydrophobic pocket which is associated with specificity.

Question 15

What is the name of the inactive form of chymotrypsin?

Answer 15

Chymotrypsinogen is produced in the pancreas.

Question 16

How is the inactive form of chymotrypsin activated?

Answer 16

Peptide bond cleavage betwen Arg-15 and Ile-16 by trypsin. This cleavage results in the pi-chymotrypsin which cleaves other pi-chymotrypsin molecules to form alpha-chymotrypsin which is the fully active version.

Question 17

What is the function of restriction endonucleases?

Answer 17

Catalyse the hydrolysis of phosphodiester backbone of DNA between 3’ oxygen and phosphorus.

Question 18

Deascribe the allosteric regulation of aspartate transcarbamoylase

Answer 18

Catalyses the first step in the biosynthesis of pyridines; the condensation of aspartate and carbamoyl phosphate to form N-carbamoyl-aspartate and orthophosphate.
Final product cystidine triphosphate (CTP) inhibits asapartate transcarbamolyase.

Question 19

Allosterically regulated enzymes do not produce the typical M-M curve instead produce a…

Answer 19

…sigmoidal curve.

Question 20

What are the two isoforms of lactate dehydrogenase and how do they differ?

Answer 20

H form in the heart has a higher affinity for substrates and high levels of pyruvate inhibit H.
L form in the liver has a weaker affinity for substrates and is not inhibited by high levels of pyruvate.

Question 21

How does phosphorylation effect protein structure?

Answer 21

Phosphoryl group adds two negative charges and the potential for three H bonds which can result in a confirmational changeor formation of a new binding site.

Question 22

Which amino acids typically under phosphorylation?

Answer 22

Ser/Thr or Tyr.

Question 23

Which amino acid is typically aceylated?

Answer 23

Lysine

Question 24

How does aceylation effect histone?

Answer 24

More acetyl groups = more transcription

Question 25

Give an example of when covalent modification is irreversible?

Answer 25

Ras and Scr become affixed to the cytoplasmic face of the plasma membrane. Lipid attachment.

Question 26

An energy source e.g. ATP is not required for proteolytic cleavage. Why is this useful?

Answer 26

It means that extracellular proteins can be activated by proteolysis.

Question 27

Is proteolytic activation reversible?

Answer 27

No

Question 28

Insulin is formed from proteolytic cleavage activation of…

Answer 28

…proinsulin

Question 29

Collagen is synthesised as soluble…

Answer 29

procollagen

Question 30

Name the five methods of regulating enzyme activity?

Answer 30

Balancing protein expression/degradation, proteolytic activation, allosteric control, covalent modification and isoenzymes.

Question 31

Describe circular dichoism?

Answer 31

Is a form of optical spectroscopy that measure the difference in absorbance between left-handed and right-handed circularly polarised light as a function of wavelength of light as it passes through an optically active sample.

Question 32

How is circular dichoism used to determine protein structure?

Answer 32

Allows for the determination of secondary structure; alpha helices 208, 222 nm and beta sheet 200 nm.

Question 33

Describe analytical ultracentrifugation?

Answer 33

Used to estimate protein size and allows protein complexes to be confirmed. Protein is removed from solution by ultracentriguation (~10^5-10^6 g). Measure sedimentation velocity as smaller proteins sediment more slowly.

Question 34

What region does Fourier-transform IR detect proteins?

Answer 34

1,700-1600 cm^-1

Question 35

Describe cryogenic electron microscopy?

Answer 35

Electron microscopy technique applied on samples cooled to cryogenic temps and embedded in an environment of vitreous water. Resolution of 3-4 A.