Unit 4

Question 1

Why would we need proteases?

Answer 1

1. Protein turnover
2. Maintain protein homeostasis (protein balance)
3. Digestion (w/ food)
4. Activation of inactivation of proteins
5. Formulation of multiple polypeptides from one polypeptide chain

Question 2

How do proteases cleave peptides?

Answer 2

By hydrolysis (addition of water)

Question 3

Why are peptide bonds stable?

Answer 3

Resonance structures reduces +ve charge on carboxyl, which reduces reactivity

Question 4

What is chymotrypsin and where can it be found?

Answer 4

A serine protease in small intestines

Question 5

What types of amino acids does chymotripsin cleave?

Answer 5

Large hydrophobic AA’s, so Trp, Phe, Met, Tyr

Question 6

How is chymotrypsin syntehsized?

Answer 6

Synthesized as cymotrypsinogen in pancreas

Activated by trypsin in small intestines to form pi-chymotrypsin, which then autolyses to form a-chymotrypsin (active)

Question 7

What is the location of the conserved serine nucleophile?

Answer 7

AA 195 (Ser¹⁹⁵)

Question 8

What is the general reaction of chymotrypsin?

Answer 8

Ser195 attacks acyl group of peptide bond, cleaving the peptide bond and forming an intermediate. Then, the intermediate is cleaved by water to release the carboxyl group.

Question 9

What is the catalytic triad contained within chymotrypsin?

Answer 9

Asp 102, His 57, and Ser 195

Question 10

How does the catalytic triad increase Ser 195’s reactivity as a nucleophile?

Answer 10

Asp 102 H-bonds with imidazole ring on His 57, making His 57 slightly positive (proton acceptor)

His 57 also H-bonds with Ser 195; His57 has acidic character and acts as a BASE CATALYST (catalyzing Ser 195 basicity)

Ser 195 thus becomes polarized from His 57 H-bonding

Question 11

What is step 1 of chymotrypsin catalysis?

Answer 11

Substrate binds to active site; hydrophobic AA in hydrophobic pocket near active site

Question 12

What is step 2 of chymotrypsin catalysis?

Answer 12

Alkoxide ion on Ser 195 (formed due to His 57 base catalysis) performs nucleophilic attack on acyl carbon, forming a transient unstable tetrahedral confirmation stabelized by Gly 195 (H bond stabelizes oxyanion tetrahedral)

Question 13

What is step 3 of chymotrypsin catalysis?

Answer 13

Tetrahedral oxyanion tetrahedral collapses, reforming carbonyl and breaking peptide bond. His 57 acts as acid catalyst, donating H to the leaving amino peptide fragment. Remaining carbonyl (C=O) stabilized by Ser 195

Question 14

What is step 4 of chymotrypsin catalysis?

Answer 14

Water enters active site and gains hydroxide-like character due to His 57 base catalysis

Question 15

What is step 5 of chymotrypsin catalysis?

Answer 15

Water’s partialy negative oxygen acts as a nucleophile (with His 57 acting as a base catalyst) forming a tetrahedral oxyanion (again stabelized by Gly 193 H-bond with oxyaninon)

Question 16

What is step 6 of chymotrypsin catalysis?

Answer 16

Tetrahedral oxyanion rearranges to form carbonyl (in carboxylic acid) ad free Ser 195. His 57 acts as acid catalyst to assist in this process.

Question 17

What is step 7 of chymotrypsin catalysis?

Answer 17

“New” carbonyl peptide leaves active site, allowing cycle to restart.

Question 18

What are similarities and differences between chymotrypsin, trypsin, and elastase?

Answer 18

Similarities: They are all serine proteases, utilizing the Serine-Histidine-Aspartate catalytic triad

Differences: Active sites are different & thus cleave different AA peptide bonds

Chymotrypsin has a large hydrophobic pocket for large hydrophobic sidechains

Trypsin has Asp at the bottom of the pocket for +vely charged AA (since + & - charge matches)

Elastase has two Valine residues that narrow down the pocket to alow the binding of small hydrophobic R groups (ex. Val, Ala)

Question 19

What are cysteine proteases? Provide an example.

Answer 19

Proteases that use cysteines as a nucleophile

E.g. caspases involved in apotosis

Question 20

What are aspartate proteases? Provide an example.

Answer 20

Ues 2 aspartate residues to act as base catalysts, such that H₂O can be used as a strong nucleophile

E.g. HIV protease

Question 21

What are metalloproteases? Provide an example.

Answer 21

Proteases that use metal ions to make water into a strong nucleophile

E.g. MMP, Matrix Metalo Protease (in cellular matrix)

Question 22

What is Hemoglobin, what is it used by, and what does it do?

Answer 22

Hemoglobin (Hb) is a protein used to carry oxygen. Used by RBC. Carries O2 from lungs (pH=7.4) to tissue (pH=7.2)

Question 23

What is the structure of Hemoglobin? Include the amino acids contained in each, as well as the secondary structures that it is made up of.

Answer 23

2x α and 2x ß subunits

α subunits have 144 AA and 7 helixes

ß have 146 AA and 8 helixes

Each subunit has one heme group (4 in total)

Question 24

How are Hemoglobin subunits bound together? Which are strong interactions, and which are weak?

Answer 24

Question 25

What is the structure of Heme? List what it is made of, and the bonds it contains.

Answer 25

Has protoporphyrin ring with Fe²⁺ in center Has 6 coordination bonds to Fe²⁺ * 4 are nitrogen * one is with histidine below ring * one is with Oxygen above ring

Question 26

Explain how O₂ bind cooperatively in heme.

Answer 26

* Fe²⁺ sticks out slightly when unbound * When O₂ binds, it "pushes" Fe²⁺ into the ring, causing confirmation change * This shifts (twists) the other α-helixes, causing increase O₂ affinity in other heme * This is called **allosteric interaction**

Question 27

What are the two states of Hemoglobin and their reltive affinities/confirmations?

Answer 27

R-state: Hb twisted, high O₂ affinity T-state: Hb untwisted, low O₂ affinity T for "tense", R for "relaxed"

Question 28

What is critical about having two Hb states?

Answer 28

Allows for high affinity for Oxygen in lungs (when you need to pick it up) and low affinity in tissue (where it needs to be released)

Question 29

What is the Bohr affect?

Answer 29

Effect of H+ and pCO₂ on Hb affinity High H+ (low pH) and pCO₂ result in LOW affinity (i.e. in tissue) Low H+ (high pH) and pCO₂ result in HIGH affinity (i.e in lungs)

Question 30

What is the biochemical explanation for the Bohr Effect?

Answer 30

a) at low pH (high [H+]) results in His residues becoming protonated, which interacts with other residues and stabelizes T-state (Low affinity "tense" state) b) CO₂ can bind to terminal amino group to form carbonate, which can interact with residues to futher stabelize T-state

Question 31

How does 2,3-biphosphoglycerate affect Hb?

Answer 31

Binding of 2,3-BPG to T-state ß subunit "pocket" stabelizes T-state (+ charge present in pocket). This decreases oxygen affinity When T→R state conversion occurs, confirmation change causes ß "pocket" to shrink in size - now too small for 2,3 BPG, it leaves, stabilizing R state and increases oxygen affinity

Question 32

Why do we need a protein around Heme?

Answer 32

1. Need protein to induce cooperativity 2. Reduces Heme affinity for CO (25000 times more affinity than O₂ for naked heme) 3. Prevents oxidation of Fe²⁺→Fe³⁺, which would result in oxygen radical formation (which is bad)