4. Protein Function and Enzymes I Flashcards
Ligands and Binding
Define the terms
ligand,
binding site,
induced fit,
substrate and
catalytic site (active site).
Ligand: bound molecules // range from small molecules to other proteins (any molecule that binds to enzyme)
Binding Site: complementary to ligand
- H-bonds, ionic interactions, van der waals interactions
- Charge complementarity
Induced Fit: enzyme’s shape and conformation changing over time in response to substrate binding
Substrate: a molecule upon which an enzyme acts
Catalytic/Active Site: a part of an enzyme to which a substrate binds to cause a chemical reaction
Ligands and Binding
Explain why ligand binding to proteins occurs transiently.
ligand binding to proteins occurs transiently because non-covalent interactions between the ligand and the protein’s binding site are relatively weak, and the protein’s conformational flexibility can influence the ligand-binding site’s shape and affinity. These factors allow ligands to associate and dissociate from proteins rapidly, allowing for the regulation of protein function and the control of cellular processes
Ligand binding to proteins occurs transiently because it is driven by non-covalent interactions, which are relatively weak and easily reversible. Non-covalent interactions include hydrogen bonding, electrostatic interactions, van der Waals forces, and hydrophobic interactions. These interactions allow ligands to associate with proteins and bind to specific binding sites, but they also allow ligands to dissociate from proteins and leave the binding site.
Ligands and Binding
List types of interactions between protein and ligand that provide binding specificity.
- Charge
- H-bonding
- Non-polar/Hydrophobic
- Shape/Size
Ligands & Binding
Interpret graphical representations of ligand binding.
see image
Ligands & Binding
Define Kd and Ka and derive equations to relate them to fractional saturation of a protein with a ligand.
- Kd = Dissociation Constant
PL ⇄ P + L
Kd =([P][L])/[PL] - Ka = Association Constant
P + L ⇄ PL
Ka = [PL] / ([P][L]) - Fraction bound:
θ = [L]/ (Kd + [L])
Kd reflects affinity
When [L] = Kd -> 0.5
↓Kd ➝ ↑affinity
Enzyme Classification
Define key characteristics of enzymes.
● Proteins (for the most part!).
● Reduce times for reactions to biological time
scales (increase kinetic rate).
● Highly Specific.
● Operate under mild conditions.
● May require cofactors.
● May be regulated
Enzyme Classification
Outline the system used in the international classification of enzymes
The nomenclature system identifies enzymes according to a unique four-digit code, the Enzyme Commission, or EC, number. The first number (the class number) corresponds to the type of reaction catalysed.
Class 1: Oxidoreductases
Class 2: Transferases
Class 3: Hydrolases
Class 4: Lyases
Class 5: Isomerases
Class 6: Ligases
Class 7: Translocases
Enzyme Classification
Name the 7 classes of enzymes and describe the type of reaction catalyzed by each class.
Class 1: Oxidoreductases
- Substrate oxidized (hydrogen or e- donor)
- Based on donor:acceptor oxidoreductase
- Dehydrogenases, oxidases, reductases
- Use NAD/FADH
- Disulfide bridge
Class 2: Transferases
- transfer a group (eg a methyl group) from one compound (donor) to another (acceptor)
- includes kinases (phosphate transfer)
Class 3: Hydrolases
- Water to cleave
- protease, collagenase, hyaluronidase
- Protein + Water -> peptide 1 + Peptide 2
Class 4: Lyases
- Cleave C-C, C-O, C-N by means other than hydrolysis or oxidation
- 2 molecules becoming one
- often named “synthases”
Class 5: Isomerases
- catalyze structural rearrangements within single molecule
- L-ala -> D-Ala
Class 6: Ligases
- add groups to molecules
- joining of two molecules with concomitant hydrolysis of the diphosphate bond in ATP (or other triphosphate)
- “synthatases”
- succinate + CoA + GTP -> succinyl-CoA + GDP + Pi
Class 7: Translocases
- Move molecules around
- movement of ions or molecules across membranes or separation within membranes
Enzyme Classification
Differentiate between ligases and lyases (synthetases and synthases).
Lyases catalyse the removal of groups from their substrate by mechanisms other than hydrolysis, leaving double bond. Whereas, ligases catalyse the linking together of compounds utilizing the energy from ATP.
Class 4: Lyases
- Cleave C-C, C-O, C-N by means other than hydrolysis or oxidation
- 2 molecules becoming one
- often named “synthases”
Class 6: Ligases
- add groups to molecules
- joining of two molecules with concomitant hydrolysis of the diphosphate bond in ATP (or other triphosphate)
- “synthatases”
- succinate + CoA + GTP -> succinyl-CoA + GDP + Pi
Active Sites and Energy Diagrams
Define the terms cofactor, coenzyme, prosthetic group, holoenzyme and apoenzyme.
PENDING
- Define the terms active site, substrate, transition state, activation energy (ΔG‡), reaction intermediate, rate-limiting step.
Active Sites and Energy Diagrams
pnding
Describe the general mathematical relationship between ΔG‡ and the rate constant (k).
Active sites and energy diagram
ΔG° is related to K by the equation ΔG°=−RTlnK. If ΔG° < 0, then K > 1, and products are favored over reactants at equilibrium. If ΔG° > 0, then K < 1, and reactants are favored over products at equilibrium
↓ ΔG‡ → ↑ k (rate constant)
Rate is related to [substrate(s)] and rate constant (k)
V= k[S]
- Describe how enzyme catalysts enhance reaction rates.
Active Sites and Energy Diagram
Enzymes enhance reaction rates by Lowering the activation energy
- Increases the rate constant and the reaction rate
↓ ΔG‡ → ↑ k (rate constant)
Catalytic Mechanisms
List mechanisms that allow enzyme catalysts to enhance reaction rates
Enzymes increase the reaction rate by:
Participating in the Reaction (directly or through
cofactors)
- General Acid/Base catalysis
- Nucleophilic/Covalent catalysis
- Metal ion catalysis
Desolvation
- Remove water, replace with polar molecule
Proximity and Orientation
- Entropy reduction
- Reacting groups near each other
- increase likelihood of Rx occurring
Stabilizing the Transition State
- “Preferential Binding” on Transition state
1.Transition state stabilization: Enzymes can stabilize the transition state of a reaction, which is the highest energy point in the reaction pathway. By lowering the energy of the transition state, enzymes can decrease the activation energy required for the reaction to occur.
1. Proximity and orientation effects: Enzymes can bring reactants closer together and in the correct orientation for the reaction to occur. This increases the likelihood of reactants colliding in a way that leads to product formation.
1. Acid-base catalysis: Enzymes can donate or accept protons to facilitate a reaction. By donating a proton to a reactant or accepting a proton from a reactant, enzymes can alter the charge distribution in the reactant, making it more likely to undergo the reaction.
1. Covalent catalysis: Enzymes can form a covalent bond with a reactant, which can lower the activation energy required for the reaction to occur.
1. Induced fit: Enzymes can undergo a conformational change upon binding to a substrate, which can facilitate the reaction by bringing catalytic residues into close proximity with the substrate.
1. Electrostatic catalysis: Enzymes can alter the electrostatic environment of a reactant, making it more likely to undergo the reaction.
1. Metal ion catalysis: Enzymes can use metal ions as cofactors to facilitate a reaction. Metal ions can stabilize charges on a substrate, donate or accept electrons, or help orient reactants in the correct orientation for the reaction to occur.
Catylic Mechanisms
Describe the role of proton transfer in reaction rate enhancement by general acid-base catalysis and identify these processes in a mechanism.
Proton transfer/donation to/from specific side chains in the enzyme active site is faster than transfer to/from solvent (H2O)
eg RNase A (ribonuclease)
(contrast “Specific acid/base catalysis” where H+ or OH- is the catalyst)
Rnase A Catalytic Mechanisms
Outline the steps in the reaction catalyzed by ribonuclease A.
(1) His 12 acts as a general base // His 119 acts as a general acid
- Promotes nucleophilic attack and bond cleavage
(2) His 12 acts as gneral acid // His 119 as general base
- promotes hydrolysis
Catylic Mechanisms
Differentiate between non-covalent catalysis and covalent catalysis and identify them in a reaction mechanism.
Covalent Catalysis:
- transient covalent bond is formed between enzyme and substrate
- alters pathway of reaction (intermediates won’t be the same)
- often involves nucleophilic catalysis
Non-covalent Catalysis
- Catalysis by neutral, organic, small molecules capable of binding and activating substrates solely via noncovalent interactions—particularly H-bonding
Catylic Mechanisms
Identify amino acid nucleophiles in a covalent catalysis mechanism.
Deprotonated amino acids: Ser, Cys, Lys, His
ROH → RO-
RSH → RS-
RNH3+ → RNH2
Catylic Mechanisms
List three ways in which metal ions may participate in reaction rate enhancement through metal ion catalysis.
(1) Binding/orientation of substrate:
- Ionic Interactions between metals and substrate can bring substrate into proximity to react, and form products.
(2) Metal ions on enzymes can stabilize the transition states of substrate.
(3) Serve as Reduction/oxidation centers:
- Metal ions can mediate oxidation-reduction reactions by reversible changes of the oxidation states of the metal
Carbonic Anhydrase is a metalloenzyme
Zinc polarizes water to stabilize OH- (nucleophile)
OH- attacks C of CO2
PEP Carboxykinase
- Magnesium and manganese metal ion cofactors help bring 2 Neg molecules together
Catalytic Mechanisms
Describe the role of metal (zinc) ions in the reaction mechanism of carbonic anhydrase.
Carbonic Anhydrase is a metalloenzyme (Lyase group 4)
- Zinc polarizes water to stabilize OH- (nucleophile)
- OH- attacks C of CO2
Class 4: Lyases
- Cleave C-C, C-O, C-N by means other than hydrolysis or oxidation
- 2 molecules becoming one
- often named “synthases”
Catalytic Mechanisms
Outline the principles of “proximity and orientation/entropy reduction in active sites.
An analogy to substrate binding.
- When the two reactive groups are spatially constrained, the reaction rate increases.
- When they are constrained in both space and orientation, that rate increases again.
- Two substrates (or the substrate and active site groups) are bound close together and in the correct orientation in the active site.
Catalytic Mechansisms
Briefly outline the reaction catalyzed by hexokinase.
Hexokinase:
Transferase (Phosphotransferase - Group 2)
- Non-covalent Catalysis
- Enzyme provides general base (Asp to remove proton)
- Mg++ stabilizes Neg Charges
- Enhanced electrophilicity of phosphate
Classic example of induced fit
- Hexokinase changes shape dramatically when glucose and ATP bind
- Brings substrates into the right geometry
- Prevents water from hydrolyzing the Phosphoanhydride
Xylose is a competitive inhibitor of Hexokinase
- Water is present when xylose is bound at thea ctive site
- Leads to “phosphorylation of water” (Hydrolysis)
Desolvation and Induced Fit
Desolvation:
- Water forms a shell around many small molecules
- Substrate binding removes many of these waters and these are replaced by interactions with polar side chains
Induced fit:
- Enzymes often change shape upon substrate binding
- Brings catalytic groups into orientation
- May be used to exclude water
Catalytic Mechanisms
Outline reasons why xylose binding to the hexokinase active site leads to ATP hydrolysis instead of phosphate transfer.
Xylose has a different shape than glucose:
- xylose binds to the hexokinase → its different shape can cause it to interact with different amino acid residues in the active site → change in the conformation of the enzyme, which can favor ATP hydrolysis over phosphate transfer.
Xylose does not form as stable a complex with hexokinase as glucose:
- Hexokinase binding glucose → stabilizes enzyme-substrate complex → facilitates the transfer of phosphate from ATP to glucose.
- Xylose does not form as stable a complex with hexokinase as glucose, which may result in a less favorable orientation of the ATP molecule for phosphate transfer.
Xylose binding may alter the electrostatic environment of the active site:
- The hexokinase active site has positively charged residues → stabilize the negatively charged phosphate group on ATP during phosphate transfer.
- Xylose binding may alter the electrostatic environment → disrupt the interactions between the active site residues and ATP and favor ATP hydrolysis instead.
Xylose binding may alter the position of the Mg2+ cofactor:
- Hexokinase requires Mg2+ as a cofactor for ATP hydrolysis and phosphate transfer.
- Xylose binding may alter the position of the Mg2+ cofactor, which could affect the ability of the enzyme to transfer phosphate from ATP to xylose. This could result in a preference for ATP hydrolysis over phosphate transfer.
Catylic Mechanisms
Use reaction coordinate diagrams to demonstrate how maximizing complementarity between enzyme and transition state (but not between enzyme and substrate) lowers net activation energy.
The difference in activation energies for catalyzed and uncatalyzed reactions (∆∆G‡cat or binding energy) is enhanced by strong interactions with the transition state, not the substrate.
∆∆G‡cat = ∆GN‡ - ∆GE‡
Catylic Mechanisms
Classify serine proteases into one of the six enzyme classes.
Hydrolase
- Proteases (peptidases)
- Cleave Peptide Bond
Serine Proteases use:
- Covalent catalysis
- General Acid/Base Catalysis
- Intermediate/transition state stabilization
Class 3: Hydrolases
- Water to cleave
- protease, collagenase, hyaluronidase
- Protein + Water -> peptide 1 + Peptide 2
Catylic Mechanisms
Explain how the substrate specificity is created for chymotrypsin, trypsin and elastase.
While the mechanisms are similar, substrate specificity is determined by the chemical nature of the “specificity pocket.”
- Chymotrypsin cleaves peptides after large hydrophobic residues. Phe/Trp/Tyr
- Trypsin cleaves peptides after positively
charged residues. (Arg/Lys)
- Elastase cleaves peptides after small, non-
polar residues (Ala)
Catylic Mechanisms
Describe the role of His57, Ser195 and Asp102 and the oxyanion hole.
Asp102: Maintains orientation and protonation state of His57 (stabilizes)
- NH has slight Pos charge = helps stabilize O- (of Ser195)
His57: General acid/base catalysis - pull H off serine
Ser195: Nucleophile (O-)
Oxyanion hole:
- Formed by backbone NH groups of residues Gly193 and Ser195,
- partially positive
- able to form hydrogen bonding interactions with a negatively-charged oxygen that develops during the reaction.
Ideal interaction is when the carbonyl carbon of the peptide bond being cleaved is tetrahedral (not planar).
Catylic Mechanisms
Draw the reactions that are involved in serine protease mechanisms.
Mechanism of serine protease.
- The catalytic triad Ser/His/Asp acts in a concerted manner and cleaves the peptide bond in two steps.
- The acyl group is firstly transferred to Ser hydroxyl oxygen, then to water.
- The dashed lines indicate favorable interactions between the negatively-charged aspartate residue and the positively-charged histidine residue, which make the histidine residue a more powerful base.
- ‘oxyanion hole’, which stabilizes via hydrogen bonding with amide N–H the negative oxygen of the tetrahedral intermediate
