CBI 6: Enzymes and Kinetics of Biocatalysis Flashcards
Give an example of an enzyme that is not a protein
- ribozymes: they are enzymes comprised of RNA
Describe this reaction profile
Describe the enzyme mechanism
- the substrate (S) binds to a specific active site of the enzyme (E), forming an enzyme-substrate complex (ES)
- interactions between the substrate(s) and the functional groups in the active site of the enzyme, lower the activation energy by providing an alternative reaction pathway
- this accelerates the reaction process as less energy is required to reach the transition state
- following the formation of ES, the reaction mechanism will form the product (P) and free enzyme (E)
- the enzyme is released and ready to bind to more substrate
How do you name enzymes?
- each enzyme is assigned four number that indicate their classification
What database can you use to view a metabolic pathway and enzymes?
- KEGG PATHWAY
Describe this graph of an enzyme-catalysed reaction without an inhibitor present
- the rate of reaction at low substrate concentrations (so the initial velocity) is directly proportional to the concentration of the substrate
- so initially first order
- at high substrate concentration, the reaction kinetics resemble zero-order kinetics
- the reaction rate is unaffected by (any further increase in) substrate concentration
What is Vmax?
- this is the maximum velocity of an enzyme
- the enzyme is still converting substrate into products but the reaction mechanism has been reached
- in an enzyme-catalysed reaction without an inhibitor, the enzyme is still converting substrate into products, but the reaction equilibrium has been reached
How do you mathematically describe an enzymatic reaction where a single substrate is converted to a single product?
- enzyme (E) combines with substrate (S) to form the enzyme-substrate (ES) complex, with a rate constant k1
- the ES has then two possible fates:
1. dissociate to E and S, with a rate constant k-1
2. proceed and form product (P) with rate constant k2 - k2 is commonly described as kcat
What is the Michaelis-Menten equation?
- When substrate concentration = KM , reaction velocity is 1/2 Vmax , meaning have the enzyme active sites are filled at that moment
Is Vmax reached?
- Vmax is approached but never reached
What does the value of KM tell us?
- it is the Michaelis-Menten constant, defined as the substrate concentration at half the maximum velocity
- it tells us about the strength of the interaction between the substrate and the enzyme
- it also reflects the specificity that an enzyme shows for a substrate
What does a small KM and a large KM tell us about the enzyme?
- Small KM:
- tells us that an enzyme binds strongly to its substrate
- needs a low concentration of substrate the catalyze the formation of the product effectively
- Large KM:
- enzyme binds weakly with its substrate
- needs a high concentration of substrate to catalyse effectively the formation of the product
Describe the Lineweaver-Burk plot
- it determines the KM and Vmax
What is kcat?
- kcat is the maximum number of chemical conversions of substrate molecules per second that a single catalytic site will execute for a given enzyme concentration
- in the equation, k2 = kcat
What does the value of kcat / KM show you?
- the value of kcat / KM (divided by) is the catalytic efficiency of an enzyme
- it combines an enzyme’s catalytic potential with its ability to bind substrate at low concentration
- therefore, the smaller the KM and the larger the kcat, the more efficient the enzyme will be
What type of bonds to reversible inhibitors have with enzymes?
- temporary, non-covalent bonds
Give the three types of reversible inhibitor and describe how they work
- Competitive Inhibitor:
- the substrate and the inhibitor molecule are similar in shape
- inhibitor binds to the active site of the enzyme
- inhibitor and substrate compete to binds to the active site
- how much inhibitor depends on the concentration of substrate
- addition of sufficient substrate can overcome the effects of competitive inhibition
- Uncompetitive Inhibition:
- the inhibitor does not bind to the unbound enzyme
- it binds once the ES complex has formed
- the release of products is prevented
- addition of extra substrate has no effect on overcoming inhibition
- Non-competitive inhibition:
- the inhibitor binds to a site that is not the active site
- substrate molecules can still bind to the active site
- inhibitor stops enzymes from performing its catalytic function
How does a competitive inhibitor affect enzyme kinetics?
Draw out the MM and LB plots
- KM will increase
- no change in Vmax
How does an uncompetitive inhibitor affect the enzyme’s kinetics?
Draw MM and LB plots
- Vmax and KM both decrease
- (think about why KM decreases)
Explain using equations below how KM is reduced with an uncompetitive inhibitor
- the inhibitor binds to the enzyme-substrate (ES) complex to form ESI
- amount of ES complex decrease
- this causes more substrate to bind to the enzyme to maintain the equilibrium
- this increase k1
- using the derived Michaelis-Menten equation, we see that if k1 increases, KM decreases
- a lower concentration of substrate is needed to formed half the maximal concentration of ES, so KM reduces
How does a non-competitive inhibitor affect Vmax and KM? And why?
Draw the MM and LB plots
- Vmax decreases
- the inhibitor stops the product from forming
- therefore, the resulting solution essentially becomes a more dilute enzyme solution
- KM stays the same:
- binding of the substrate is not affected by the inhibitor so KM is not affected
What are cofactors?
- some enzymes require non-protein chemical or metal compounds attached to them to work
- they assist reaction
- usually coenzymes (small, organic, vitamin-derived molecules such as FAD) or inorganic metal ions
How does pH affect enzymes?
- the rate of enzyme-catalysed reactions depend on the pH of the solution
- each enzyme has an optimal pH
How does temperature affect enzyme activity?
- every enzyme has an optimal temperature
- generally increasing temperature increase rate of enzyme activity up to its optimum temperature
- higher temperatures would denature the enzymes by causing the molecules to vibrate and twist so rapidly that their non-covalent bonds break
- if a protein loses its secondary and tertiary structure, it becomes denatured
Describe how reversible covalent modification can help regulate enzymes involved in signal transduction
Example:
- an enzyme can be phosphorylated and activated/deactivated by another kinase enzyme
- this type of regulation is reversible
- phosphatase enzymes are able to remove the phosphate group
- other common modifying groups are acetyl-, methyl-, carboxyl-
Define allosteric inhibitor
- an agent that decrease enzyme activity by binding an allosteric site
- allosteric binding causes a conformational change in the enzyme that can result in competitive, non-competitive, or uncompetitive inhibition (or a combination of these effects)
Are non-competitive inhibitors the only type of allosteric inhibitors?
- no
- all non-competitive inhibitors are allosteric inhibitors but not all allosteric inhibitors are non-competitive
How could allosteric binding cause competitive and uncompetitive inhibition effects?
- binding to the allosteric site could cause a conformational change in the active site, preventing substrate binding or obscure/prevent sufficient access to it
- causing competitive inhibitory effects
- allosteric binding could stabilise an enzyme-substrate complex
- resulting in uncompetive inhibition
Does allosteric binding always result in inhibition?
- no, it can also result in the activation of an enzyme
What does the sigmoid graph of reaction velocity against substrate concentration display?
- most allosterically-regulated enzymes are proteins with a quaternary structure
- multisubunit allosteric enzymes show sigmoid shapes
- their multiple subunits and their allosteric sites mean that they do not obey Michaelis-Menten kinetics and cannot be differentiated by analysis using Lineweaver-Burk plots
Describe the shape of the sigmoid graph
- the reaction gradually increases as substrate concentration increases
- after the first active site is filled, other substrates bind are more likely to bind to the other active sites
- this is a due to a quaternary change to the enzyme’s structure
- this increases the reaction velocity
- as all the sites are saturated, reaction rate reaches a plateau
Why are allosteric enzymes very important in regulating metabolic pathways?
- they are very sensitive to relatively small changes in substrate and also inhibitor concentration
