Lecture 11: Protein Structure & Function Part 3 - Enzymes Flashcards
1
Q
Enzymes
A
- Catalytic proteins that speed up cellular reactions to allow life
- Most are catalytic proteins, but there are catalytic RNAs called ribozymes
- Don’t affect ΔG
- They cannot make a reaction occur spontaneously if it’s thermodynamically unfavorable
- They cannot alter the concentration of reactants and products in a reaction mixture that is at equilibrium
- They cannot not extract more useful energy per mole of reactants - they can only extract it faster
- Determine the rates of nearly all the chemical transformations that make or break covalent bonds in cells
- They reduce the activation energy in a reaction
- The energy of the reactants and the products are the same as without a catalyst
- Enzymes don’t affect thermodynamics of reaction
2
Q
Enzymes as catalysts
A
- Is only required in small amounts
- Must be left unchanged at the end of a reaction, so that it can cycle back to bind more substrate
- Catalyzes equally the forward and reverse reactions
- Can increase the rate of a reaction by 10^8 to 10^12 fold
3
Q
Enzyme-substrate binding
A
- Enzyme function begins by binding the substrate (S) through reversible, weak bonding to a stereo-specific active site (i.e. 3D-shape of substrate matters)
- This forms an enzyme-substrate complex
- Substrate is chemically converted to product, forming an enzyme-product complex
- Finally, the product is released and the enzyme can bind another molecule of substrate
4
Q
Enzyme Kinetics
A
- Vmax = rate when enzyme is saturated with substrate
- KM = “Michaelis constant”; equal to the dissociation constant for the enzyme-substrate complex; when reached, it accounts for half the Vmax
- Low KM = enzyme has a high affinity for its substrate
- In general, the value of the KM of an enzyme lies within its substrate’s natural concentration range
- If KM is too high or too low, the rate of the reaction won’t change very much and the cell won’t be able to respond to changes in substrate concentration
- Rate = Vmax × [S] / (KM + [S])
5
Q
What does enzyme binding do?
A
- Substrate molecules must pass through a series of intermediate states in which:
-> 3D geometry of reactants needs to be adjusted for optimal interaction
-> Electrons among reacting atoms must begin to become redistributed
Intermediate states have higher energy than reactants or products, so they are unstable
-> There can be several intermediate states
A + B —> A—B (unstable transition state) —> C + D
6
Q
Transition states
A
- An intermediate term between product and reactant, with a specific structure
- Enzymes function by stabilizing specific transition states of the structure (lowering their energy)
- > As they react, substrates will go through a start with a higher energy level before the reaction occurs
- > This state has higher energy than either reactants or products
- > The energy needed to reach it is the major part of the activation energy for the reaction
- > Enzymes stabilize transition states and thus lower activation energy, which speeds up the reaction
7
Q
What are mechanisms that enzymes use to stabilize transition states?
A
1) The enzyme binds to two substrate molecules and orients them precisely to encourage a reaction between them
2) Binding of the substrate to the enzyme rearranges electrons in the substrate, creating partial negative and partial positive charges that favor a reaction
3) The enzyme strains the bound substrate, forcing it toward a transition state to favor a reaction
8
Q
Prosthetic groups/Cofactors
A
- Non-protein molecules which aid in some protein function
- > They can be covalently and non-covalently bound by the protein
- > Hemoglobin uses heme as a prosthetic group for oxygen transport
- Are referred to as cofactors (e.g. Mg+2 is a critical cofactor for enzymes that join or cleave nucleic acids)
9
Q
Coenzymes
A
- Organic molecules that act as cofactors
- > Vitamins are frequently these or their precursors
- > All these are cofactors, all cofactors are prosthetic groups, but not all prosthetic groups are these
10
Q
Molecular tunnels
A
- Some enzymes perform multiple sub-reactions, that must occur at distinct active sites
- Structures of such enzymes many act as tunnels to direct the intermediate products from one active site to the next - the intermediates never leave the enzyme
- This prevents diffusion of intermediates, prevents decomposition of unstable molecules and speeds up reaction rates
- Carbamoyl phosphate synthetase: 3 active sites connected by molecular tunnels
11
Q
Multi-enzyme complexes
A
- Most metabolic pathways require reactions to occur in specific, highly regulated order with different enzymes
- Enzymes in the pathway may be organized into higher-order multi-enzyme complexes (non-covalently associated)
- > Multi-enzyme complexes are not the same as multi-subunit enzymes
- > In multi-enzyme complexes, each enzyme can function independently
- The product of the 1st enzyme is passed to the 2nd enzyme, where it’s the substrate and so on
- This prevents diffusion of the products, and allows for coordinated regulation of the pathway
- > Being part of a complex, metabolic pathways can function more efficiently
12
Q
Enzymes are tightly regulated
A
- Enzymes must be able to respond rapidly to changing cellular and extracellular conditions
- Many enzymes aren’t constitutively active, and so must be turned on when needed (and off when not needed)
- Regulation of key enzymes often involves multiple inputs - back to the idea of molecular integrators
- > Enzymes need to be able to recognize multiple inputs in order to be regulated efficiently
13
Q
Feedback Control
A
- When a downstream product regulates an upstream enzyme in a given pathway
- Mostly negative feedback, but sometimes positive feedback
- Negative feedback is when one of the products of a reaction will turn off an enzyme after enough is made
- ATP is a substrate of phosphofructokinase, a key enzyme in glycolysis, but the concentration of ATP that allows it to function is low, so when there’s high levels of ATP, phosphofructokinase shuts off
- This happens because there’s 2 ATP binding sites on phosphofructokinase
14
Q
Irreversible inhibitors
A
- Covalently binding to an amino acid residue - usually lower Vmax by effectively inactivating and “removing” active enzyme molecules
- Rare in nature (because energetically costly to undo), but not in industry [Aspirin (acetyl salicylic acid) is an irreversible inhibitor of enzymes COX-1 and COX-2, by acetylating a serine in the active site
15
Q
Competitive reversible inhibitors
A
- reversibly bind to active site and compete with substrate; can be displaced by very high [S]
- Increase KM = lower “affinity”
- Doesn’t reduce Vmax
