MT 2 Flashcards
(163 cards)
Chymotrypsin:
[enzyme type] in [organ]
Can cleave [?] bonds on the [?] terminal side of [?] amino acids [give 4 examples]
Synthesized as [precursor] in the [organ]; it is activated in the [organ] by [?] first, then by [?] in a process called [?]
Active chymotrypsin is composed of [#] chains, connected together by [?] bonds
In the active site, it has conserved reactive [?] that can act as a [?]
Serine protease in small intestines
Can cleave peptide bonds on the carboxy terminal side of large hydrophobic amino acids (ie. Trp, Phe, Tyr, Met)
Synthesized as chymotrypsinogen in the pancreas; it is activated in the small intestine by trypsin first, then by itself (autolysis; π-chymotrypsin cleaves itself)
Active chymotrypsin is composed of 3 chains, connected together by disulfide bonds
In the active site, it has conserved reactive Ser (S195) that can act as a strong nucleophile
Protease
Enzymes that break peptide bonds
Cleave the peptide bond by hydrolysis (addition of water)
What is the purpose of a protease? (5)
- Protein turnover (ie. reuse proteins)
- Maintaining protein homeostasis (ie. degrading misfolded proteins)
- Digestion
- Activation or deactivation of proteins (enzymes)
- Formation of multiple proteins from one polypeptide chain
Why are peptide bonds stable?
Resonance structures
The carbonyl is less electrophilic due to resonance structures, so it is less reactive compare to regular carbonyl groups.
Overview of Mechanism of Serine Proteases (2 steps)
- The nucleophilic oxygen on serine attacks the electrophilic carbonyl of the peptide bond in a nucleophilic attack, cleaving the peptide bond and covalently forming an acyl-enzyme intermediate.
- The intermediate is cleaved by water to generate the new carboxyl group and free enzyme (S195 is regenerated)
Why is S195 so reactive?
S195 forms H-bond with imidazole ring of His57
This H-bond positions S195 and polarizes the hydroxyl group of S195 (it has alkoxide character)
His acts as a base catalyst (pulls away H+ from S195)
Asp102 also H-bonds with His57, making it a better proton acceptor
Asp102, His57, and Ser195 are known as catalytic triad (true for all serine proteases)
Step-By-Step Catalysis by Chymotrypsin
- Substrate binds to the active site; large hydrophobic amino acids fit to the hydrophobic pocket next to the active site
- The alkoxide ion on S195 does nucleophilic attack on the carbonyl carbon of the substrate. This causes carbon to transciently adopt a tetrahedral geometry. His57 acts as a base catalyst
- The tetrahedral intermediate rearranges to the acyl-enzyme intermediate. The carbonyl reforms and the peptide bond is cleaved. His57 acts as an acid catalyst, donating proton to the amine group. The “new” amino peptide fragment leaves the active site.
- Water molecule enters the active site; His57 H-bonds with water, making it hydroxide-like
- The nucleophilic oxygen of water acts as a nucleophile, attacking the carbonyl carbon. His57 acts as a base catalyst, stealing H from water. This forms another short-lived intermediate– tetrahedral oxyanion
- Tetrahedral intermediate rearranges, reforming the carbonyl and separating the product from Ser195. His57 acts as an acid catalyst (H donor to Ser195). A “new” carboxyl peptide is formed and free Ser195 are generated.
- The “new” carboxyl peptide leaves active site. Enzyme is ready to catalyze another reaction.
What are some examples of serine proteases? How do they differ from each other?
Serine proteases all use the same catalytic mechanism (ie. same catalytic triad), but they all have different specificity
- Chymotrypsin has a large hydrophobic pocket, so it binds amino acids with large, hydrophobic side chains
- Trypsin has Asp at the bottom of the pocket, so it binds positively charged amino acids
- Elastase has 2 Val residues that narrow down the pocket, so it binds amino acids with small R-groups (ie. Ala, Val)
List 4 types of proteases
- Cysteine proteases
- Aspartate proteases
- Metalloproteases
- Serine proteases
Cysteine proteases
Use cysteine as a nucleophile
Example: caspases in apoptosis
Aspartate proteases
Have two aspartates that make water a strong nucleophile
Example: HIV proteases
Metalloproteases
Use metal ion to make water a strong nucleophile
Example: MMP - matrix metalloproteinase; regulates tissue remodelling
Hemoglobin and Oxygen Transport
Hb Oxygen is soluble in water, but not enough to supply tissues
RBC (red blood cells) use Hb to carry oxygen from lungs (high [O2], low [CO2], pH=7.4) to tissues (low [O2], high [CO2], pH=7.2) Carry O2 from high [O2] to low [O2}
Hemoglobin structure
Tetramer (quaternary structure) - 2 alpha and 2 beta subunits
Alpha subunit - 144 amino acids; 7 helices
Beta subunit - 146 amino acids; 8 helices
a1β1-a2β2 interface is weaker, has fewer interactions and is less rigid
The other interface (between α1-β1 and β2-α1) is very stable and has many interactions
Each subunit has 1 heme group (4 in total); the O2 binds to heme
Heme has protoporphyrin ring with Fe2+ in the centre Fe2+ has 6 coordination sites (4 are nitrogens from the ring; 5th is from His below the ring; 6th is above the ring and binds oxygen)
How does heme bind O2?
When the 6th site is empty, the iron is a little outside the ring
When it binds to O2, it “pops” into the ring, because the electron density changes
The histidine (at the 5th site) is attached to a-helix, so in turn, the a-helix moves, causing carboxyl end to move
This disrupts and creates new ion pairs in the α1β1-α2β2 interface
This conformational change increases the other hemes ability to bind O2
H6 binds oxygen cooperatively
H6 with 3 O2 bound has a 20x greater affinity to O2 compared to when no O2 is bound – this is known as allosteric interaction (ie. change in shape of a protein that results form binding a molecule at a point other than the active site)
What are the two states of hemoglobin?
The untwisted deoxyHb (low O2 affinity) = T-state
Twisted oxyHb (high O2 affinity) = R-state
How does cooperativity help O2 transport?
If Hb was always in R-state, it would bind O2 in lungs but never release it in the tissues
If Hb was always in T-state, it would bind only small amounts of O2 in the lungs, which is not enough for metabolism
Because Hb binds oxygen cooperatively, it can exist in R-state in the lungs and become saturated with O2, and then travel to the tissues where it can convert to T-state and release O2
Why does it convert from R-state to T-state? List the two main ideas.
- Bohr effect
- 2,3-bisphosphoglycerate (2,3-BPG)
What is the Bohr Effect?
H+ and CO2 promote the release of O2 from oxyHb, lowering affinity
This enhances Hb’s ability to release O2 in the tissues and pick it up in the lungs
What is the [H+] and [CO2] in the tissues and lungs?
[H+] and [CO2] is high in the tissues, resulting in low affinity to oxygen due to the Bohr effect.
[H+] and [CO2] is low in the lungs, resulting in high affinity to oxygen due to the Bohr effect.
What is the mechanism of the Bohr effect?
H+ : some His residues in the α1β1-α2β2 interface have pKa’s close to 7. In conditions of low pH (high [H2]) the histidines become protonated and will interact with other residues, stabilizing the T-state
CO2 : can bind to the free terminal amino group; carbonate ions can form new ionic interactions that will stabilize the T-state
How does 2,3-bisphosphoglycerate impact the conversion of R-state to T-state?
DeoxyHb binds to 2,3-BPG, which stabilizes the T-state, lowering affinity to O2 It binds in a special pocket formed by the 2 β subunits
The pocket has a positive charge to interact with the negatively charged 2,3-BPG
Part of transition from T-state to R-state is the removal of 2,3-BPG from Hb
Conformational changes during T→R make the pocket too small for 2,3-bBPG to fit; thus, 2,3-BPG leaves, stabilizing the R-state.
The binding curve shifts, resulting in higher affinity.
Why do we need the protein part of hemoglobin? Why don’t we just have heme?
- Cooperativity (only works with protein)
- CO binds to heme 25,000x better than O2; Hb lowers the affinity for CO by shielding heme
- Prevents iron (Fe2+) from being oxidized (to Fe3+) because the protein has interactions that shield iron from being oxidized
Metabolism
Highly integrated network of reactions (chemical pathways) that enable a cell to extract energy from the environment and use this energy for biosynthesis
