Module 2 Flashcards
(28 cards)
What are the three main outcomes of a protein in the cell?
1) They fold correctly without help
2) They fold correctly with the help of a chaperone protein
3) Misfolded proteins are recognized for degradation
What are common examples of molecular chaperones (heat-shock proteins) and where are they found?
Hsp70: cytosol, mitochondria
BiP: endoplasmic reticulum
DnaK: bacteria
Hsp70 Pathway (Molecular Chaperone)
1) Hsp70 has a nucleotide-binding domain for ATP and a substrate-binding domain for unfolded or nascent proteins
2) Hsp70 binds to the unfolded protein via hydrophobic regions, preventing misfolding
3) ATP hydrolysis, assisted by DnaJ/Hsp40, allows the protein to fold properly
4) ADP released, assisted by GrpE/BAG1
5) New ATP binds, Hsp70 is reset
T or F: Molecular chaperones are monomeric proteins, whereas chaperonins are multimeric large macromolecular complexes
True
What are common examples of chaperonins and where are they found?
Hsp60: mitochondria
TCiP: eukaryotic cytosol
GroEL: bacteria or chloroplast
GroEL Pathway (Chaperonin)
1) Misfolded protein enters one of GroEL’s two chambers, binding to the hydrophobic inner surface
2) 7 ATP bind to GroEL, triggering GroES to cap the chamber, sealing the protein inside
3) Chamber enlarges, protein folds properly
4) ATP hydrolysis allows the GroES cap to come off and the protein is released
5) GroEL is ready again, the next protein will go into the opposite chamber
6) GroEL is composed of 7x Hsp60 subunits, which each require ATP to activate
Protein Degradation is a Two-step Process
1) Protein is tagged by ubiquitin
2) Proteasome cleaves the target protein into short peptide sequences (7-8 residues)
Ubiquitinylation Has a 3 Enzyme System
E1: Ubiquitin activating enzyme, picks up free ubiquitin in the cytosol
E2: Ubiquitin conjugating enzyme, attaches ubiquitin to the target protein
E3: Ubiquitin Ligase, recognizes the specific target protein for degradation
Ubiquitinylation Pathway
1) E1 attaches to free-floating ubiquitin, which requires ATP hydrolysis
2) Ubiquitin is transferred to E2
3) E3 captures and recognizes the target protein
4) Ubiquitin is transferred from E2 to E3
5) Poly-ubiquitinylation: multiple ubiquitin are added
Proteasome Degradation Pathway
1) Polyubiquitin tag is recognized by the cap of the proteosome
2) Target protein unfolds as it enters and is cleaved into small peptides
Spinocerebellar Ataxia
A disease that results from a mutation in the Ataxin 1 gene, creating a misfolded Ataxin protein. This is lethal because the misfolded protein is still tagged with ubiquitin, but cannot be unfolded by proteosomes, resulting in aggregation, and proteosomes being unable to degrade other necessary proteins
What are the two main factors that determine protein-ligand binding?
Specificity (selective binding) and Affinity (binding strength between protein and ligand)
What is molecular complementarity?
The precise fit between a protein and its ligand, allowing noncovalent interactions
Explain the difference between the association constant (Keq) and the dissociation constant (Kd)
A high association constant (Keq) or a low dissociation constant (Kd) indicates a high-affinity interaction where the protein and ligand bind, and vice versa
T or F: In reference to enzyme kinetics, a graph with concentration of a substrate can be plotted against the rate of reaction. Vmax is the maximal velocity or rate of reaction that can occur. If the concentration of enzyme is the same, whether it’s a high-affinity substrate or low-affinity substrate, Vmax will be the same. However, it’ll take a low-affinity substrate a higher concentration of substrate to reach the same rate of reaction as a high-affinity substrate.
True
Michaelis Constant (Km)
The concentration of substrate at which reaction velocity is half maximal (half of Vmax). A substrate with a higher Km value has a lower affinity to that enzyme
T or F: In a scenario where the substrate remains the same but there are varying degrees of enzyme concentration, Vmax indeed changes but Km remains the same. With more enzyme available, the reaction is faster but Km is the same nonetheless.
True
Protein Kinase A Pathway
1) Protein Kinase A (PKA) is a kinase enzyme that adds a phosphate group to a protein.
2) Two substrates bind, the target protein and ATP.
3) A small domain and large domain contribute to forming binding sites for both substrates.
4) They bind when PKA is in open conformation, which causes a change in shape as the glycine lid traps the substrates inside, allowing the transfer of a phosphate from ATP to a protein
5) Since both substrates changed in structure, they no longer have high affinity for those binding sites, allowing them to leave
6) Molecular complementarity regulates activity, as only ATP binds, and the large domain has glutamic acid residues that recognize specific sequences of the target protein (Arg & Ser).
5 General Mechanisms for Regulating Protein Function
1) Allosteric Regulation
2) Signal-Induced Regulation of Protein Levels
3) Covalent Modification
4) Proteolytic Cleavage
5) Enzyme Complexes
PKA Allosteric Activation Pathway
1) Inactive PKA is a tetramer that contains two regulatory subunits and two catalytic subunits
2) In the inactive state, PKA’s substrate binding site is blocked by a pseudo-substrate
3) cAMP is an allosteric activator of PKA, as it binds to the regulatory subunits, the pseudo-substrates release, allowing these enzymes to activate
ATCase Allosteric Inactivation Pathway
1) ATCase has six catalytic and six regulatory subunits
2) CTP is an allosteric inhibitor
3) CTP bind to regulatory subunits, causing the entire complex to twist into an inactive tense conformation
4) When CTP is low in concentration, the ATCase enzyme is in relaxed conformation and active (can bind to aspartate)
5) If the concentration of CTP is high, it acts as a negative modulator for itself, preventing the cell from producing more
T or F: In the case of ATCase, CTP acts as an allosteric inhibitor and ATP acts as an allosteric activator. Since there is a constant concentration of enzymes, Vmax remains the same. However, ATCase with ATP has a lower Km, a higher affinity for aspartate. In contrast, ATCase with CTP has a higher Km, a lower affinity for aspartate.
True
Co-operative Allostery
1) A specialized form of allosteric modulation where the binding of a ligand to one subunit in a multimeric complex, leads to the change in affinity of all subunits
2) For an allosteric enzyme to reach Vmax, only a small concentration of ligand is required, producing a sigmoidal curve (S-shaped curve)
What is 2,3-BPG?
1) A molecule found in high concentrations in targets that receive oxygen from hemoglobin
2) It decreases the affinity of hemoglobin for oxygen
3) Fetal hemoglobin has a higher affinity for oxygen, so it also has a lower affinity for 2,3-BPG
