Chapter 8 Flashcards
What effect do enzymes have on the rate of a reaction and the equilibrium that is reached?
The effect enzymes have on the rate of a reaction is that they accelerate reactions. Enzymes accelerate the attainment of equilibria but do not shift their positions. The eqilibirum position is a function only of the free-energy difference betwee reactants and products.
What are the characteristics of Enzyme Specificity/Catalytic Power?
- Catalysis takes placeat a particular site on the enzyme called the active site. Enzymes catalyze reactions by stabilizing transition states, the highest-energy species in reaction pathways .
- Proteins are highly effective catalysis for an enormous diversity of chemical reactions because of their capactiy to specifically bind a very wide range of molecules.
- Enzymes are highly specific both in the reactions that they catalyze and in their choice of reactants (substrates)
- Function of enzymes: accelerate the rate of reaction. An enzyme will either SPEED things up or START things up. Most reactions in biological systems do not take place at perceptible rates in the absence of enzymes.
An example would be: A proteolytic enzyme which can catalyze proeolysis (the hydrolysis of a peptide bond)
Protease: a protein that breaks peptide bonds.
It can form an [ES] compound (enzyme substrate)
The specifictiy of an enzyme is due tot he precise interaciton of the substrate with the enzyme… the precision is a result of the intricate 3D strucutreof the enzyme protein.
How do proteolytic enzymes differ?
Proteolytic enzymes differ markedly in their degree of substrate specificity:
- Subtilizin is an enzyme that cleaves almost any peptide bond.
- Thrombin cleaves between arginine and lysine. Most SPECIFIC and blood clotting.
- Trypsin: it’s job is to digest peptide bonds in the food we eat to break down into amino acids Trypsin cleaves to the hydrolysis site. Cleaves peptide bonds only on the carboxyl side of lysine or arginine. Positively charged side chains (bases)
What other characteristics of enzymes?
Many enzymes require cofactors for activity.
Cofactors can be…
- Metal ions
- coenzymes (small organic molecule)
- lose binders … act like a substrate: bind at an active site participate in the reaction then dissociate
- Tight binders… alwasy at the active site, do not dissociate this is referred to as a prosthetic groups
Define the terms apoenzyme, holoenzyme and prosthetic group.
1. Apoenzyme: An enzyme without its cofactor
If you have the globin but not the heme (hemoglobin will not work. It is inactive
2. Holoenzyme: Complete, catalytically active enzyme
Ex. Globin + hehe = hemoglobin = holoenzyme which is active
3. Prosthetic Group: tightly bound coenzyme
What is Free Energy and how is it affected by enzymes?
Free enery is the difference in free energy from substrate and the product.
- Enzymes only affect the energy required to initiate the conversion of reactants into products (determines the rate of the reaction)
- The free energy change of a reaction tell us if the reaction can occur spontaneously…
- If delta G is negative a spontaneous reaction (exergonic) doesn’t necessarily need an enzyme to occur.
- If delta G is equal to Zero no net change in that reaction will occur. the reaction has reached equilibrium.
- If delta G is positive a non-spontaneous reaction (endergonic) requires enzyme and an input of free energy is required to drive such a reaction.
What can we tell about an enzymatic reaction if we consider the change in free energy that accompanies the reaction?
The free energy of a reaction will decrease if an enzymatic reaction is occurring. To understand how enzymes operate, we need to consider only two thermodynamic properties of the reaction:
- The free-energy difference (delta G) between the products and reactants
- The energy required to initiate the conversion of reactants into products.
The former determines the rate of the reaction will take place spontaneously, whereas the latter determines the rate of the reaction. Enzymes affect only the latter or the rate of the reaction.
What effect does an enzyme have on the ability of a transition state to be formed and teh corresponding GIbbs free energy of activation?
The free-energy difference between reactant and products accounts for the equilibrium reaction but enzymes accelerate how quickly this equilibirum is attained.
Free energy decreases as a consequence of the reaction, must release this energy.
Binding energy: free energy that is released as a consequence of substrate binding. To form the transition state.
The transition state: the least stable form. The fleeting molecule and has a higher free energy than the substrate or product.
Enzymes facilitate the formation of the transition state.
Enzymes accelerate reaction by decreasing delta g, the activation energy.
Describe the enzymes and how it affects the rate…
Enzymes alter the rate of the reaction but they have no affect on the equilibrium that is reached.
The same equilibrium point is reached but much more quickly in the presence of an enzyme.
The Kf = forward rate constant and Kr = reverse rate constant.
The equilibrium constant = ratio of these rate constants
Therefore, you will see 100x more A than there is B… the equilibrium concentration of P is 100 times that of S, whether or not enzyme is present.
With enzyme… reach equilibrium in 1 second
No enzyme reach equilibrium in 1 hour.
Gibbs free energy of activation:
The difference in free energy between transition state and substrate
Function of enzyme is to decrease this free energy… facilitate teh formationof the transition state.
Increases teh likeliness that the reaction will occur.
the combination of the substrate adn enzynme creates a reaction pathnway whose transition state energy is lower than that of the reaction in the absence of enzyme.
The speed of the reaction will be dependent on the ability to form the transition state.
- Easy to form a transition state its a fast reaction
- Difficult to form a transition state it is a slow reaction.
Concentration of enzyme is fixed/constant.
Maximum velocity: the fastese that enzyme can catalyze product formation. It is determined by how easy/hard the transition state is formed. All the catalytic sites are filled adn so the reaction rate cannot increase (if you’re driving your car and you have your gas pedal to the floor thats as fast as youre going to go)
Hyperbolic curve:
- Velocity is proportional to substrate concentration. Double substrate concentration = double number of active sites.
- AT the top of the curve, velocity is independent of substrate concentration.
What are some of the common features that govern the function of an active site of an enzyme?
The acitve site of an enzyme is the region that binds the substrates. Also it contains the residues that directly participate in the making and breaking of bonds. The interaction of the enzyme and substrate at the active site promotes the formation of the transition state. The common features of the function of the active site include:
- the active site is a 3D cleft, or crevice formed from different parts of the amino acids.
- The active site takes up a small part of the total volume of an enzyme
- Active sites are unique microenvironments
- Substartes are bound to enzymes by multiple weak attractions
- The specificity of binding depends on the precisely defined arrangement of atoms in an active site.
There are different ways for enzyme and substrate to bind. There is the lock adn key model and the induced fit model.
What is an active site?>
Active Site: the region on an enzyme that binds the substrates.
_1. The active site is a 3D cleft or crevice formed by groups taht come from different parts of the amino acids sequence. _
Active sites usually located in some sort of cleft/crevice in that protein structure
Located on surface = more difficult to be more specific, but putting it into cleft/ crevice allows it to filter things taht may react at active site (makes it more specific)
2. The active site takes up a relatively small part of the total volume of an enzyme
6 amino acids here to regulate and form the acitve sites of the enzyme
Active site formed from small number of amino acids
location of amino acids are not located near each other within the primary sequence.
3. Acitve sites are unique microenvironments
Non-polar microenvironment of the cleft enhances the binding of substrates as well as catalysis
Water is excluded from the active site (unless it is a substrate) … b/c water is a polar molecule and this will cause trouble due to electrostatic interference.
4. Substrates are bound to enzymes by multiple weak interactions
_5. The specificity of binding depends on the precisley define arrangement of atoms in an active site. _
What is the Lock-and-key model of enzyme substrate binding?
The active site of the unbound enzyme is complementary in shape to teh substare
What is the induced-fit model of enzyme-substrate binding?
The enzyme changes shape on substrate binding. The active site forms a shape complementary to the substrate only after the substrate has been bound.
What is lysozyme?
It helps break down the cell wall
What does Km mean? How does one determine the Km and Vmax for an enzymatic reaction?
Km is Michaelis constant. It has the units of concentration and is independent of enzyme and substrate concentrations. It is an important characteristic of enzyme-substrate interaction.
Km = (k-1 + k2) / k1
Vmax = k2 [E]T
Km values of enzymes range widely and depend on the particular substrate and on environmental conditions such as: pH, temperature, and ionic strength of buffer used.
Km is a characteristic of individual enzymes
Km is the amount of enzyme needed to reach half-max velocity
Another way to characterize enzymes is by looking at rate constans.
The ES complex dissociates into E and S much more rapidly than product is formed. Km is equal to the dissociation constant of the ES complex if k2 is much smaller than k1. When this condition is met Km is a measure of the strength of the ES complex (inversely proportional)
- If km is low –> high affinity (strong) binding
- if km is high –> high affinity (weak) binding
What are sequential displacement mechanisms?
Sequential Displacement
- All substrates must bind to the enzyme prior to the release of any products
- In a bisubstrate reaction, a ternary complex of enzyme and both substrates forms.
Sequential ordered mechanisms
- Substrates will bind in a particular order
- NADH will always bind first followed by pyruvate
- Products will be released in a particular order
- Lactate will be produced first followed by NAD+
- At the end of the reaction, the enzyme has to be reproduced.
Sequential ordered mechanisms:
- “random” = order does not matter
- ATP or creatine may bind first
- ADP or phosphocreatine may be released first.
- The formation of phosphocreatine and ADP from ATP and creatine, catalyzed by creatine kinase
Describe the double displacement mechanism (ping pong)
- One or more products are released before all substrates bind the enzyme.
- Defining feature: substituted enzyme intermediate –> enzyme is temporarily modified
- “ping pong” –> substrates appear to bounce on and off the enzyme analogously
- Substrate 1 binds and forms an ES complex and Product 1 is released. Part of substrate is left behind –> modified.
What are the characteristics of Competitive inhibitors?
- The competitive inhibitor oftne resembles the substrate and binds to the active site of the enzyme and prevents product formations.
- Diminishes the rate of catalysis by reducing the proportion of enzyme moleucles bound to a substrate
- Can form EI complex or ES complex but NOT and EIS complex
- Therefore only one thing can bind at the same time
What will determine whether I or S binds?
- Concentration: competitive inhibition can be relieved by increasing the substrate concentration.
- Affinity: Ideally when designing a drug you want to creat drugs that have high affinity.
There is also such thing as a reversible competitive inhibitor.
Competitive inhibition
Reversible compeitive inhibitor
As we increase inhibitor concentration, K increases
Can always overcome the affects of the inhibitor by adding more substrate
Reversible and Vmax shifts to the right
Competitive Inhibition
Km is increased and there is no chagne in vmax
What are the characteristics of non-compeitive inhibitors
- Does not interact with the active site; has its own binding site
- Cannot be overcome by the addition of more substrate
- No structural requirements
- Inhibitor and substrate bind simultaneously to an enzyme molecule at different binding sites
Can form:
- EI –> no product
- ES –> product
- EIS –> No product
When you have the inhibitor bound, substrate cannot be converted to product!
Acts by decreaseing the turnover number rather than by diminishing the protportion of enzyme molecules that are bound to substrate
Even if you continue to add substrate, the curves with non-competitive inhibitor will never reach Vmax.
As we increase the concentration of our inhibitor more and more molecules of enzyme will be inhibitor bound.
Every enzyme that is inhibitor bound will be blocked from producing any product (even if they are substarte bound)
Enzyme molecules that don’t have inhibitor bound act just like they did normally
Non competitive inhibitor
No change in Km
Drcrease in Vmax
