Topic 11: Enzyme kinetics and regulation Flashcards
Name the 6 types of enzymes and their functions
Oxidoreductases are enzymes that catalyze the transfer of electrons from one molecule, the reducing agent, also called the electron donor, to another, the oxidizing agent, also called the electron acceptor
Lactate is the substrate, pyruvate is the product, and NAD+/NADH is the cofactor
Transferases catalyze the transfer of a group of atoms, such as amine, carboxyl, carbonyl, methyl, acyl, glycosyl, and phosphoryl from a donor substrate to an acceptor compound.
In the example, L-alanine and alpha ketoglutarate are the substrates, and pyruvate and l-glutamate are the products
Hydrolases are the group of enzymes that catalyze bond cleavages by reaction with water (hydrolysis)
In pyrophosphatase, pyrophosphate/H2O are the substrates, and phosphate is the product
Lyases or synthases catalyze the breakdown of chemical bonds through methods other than hydrolysis or oxidation. They differ from other enzyme classes in that most reactions catalyzed by lyases only require one substrate molecule for the forward reaction, and two for the reverse reaction
Isomerases catalyze changes within one molecule. They convert one isomer to another, meaning that the end product has the same molecular formula but a different physical structure
Ligases (synthetases) are enzymes that are capable of catalyzing the reaction of joining two large molecules by establishing a new chemical bond, generally with concomitant hydrolysis of a small chemical group on one of the bulky molecules or simply linking of two compounds together
Requires ATP due to forming bonds, so often ATP acts as a cofactor
What are some ways enzyme activity can be measured?
Detection of products: If a product is colored, a spectrophotometer can be used to observe how much product is formed through recording absorbance
Ex: Caspase 3 catalyzes the hydrolysis od DeVD-pNA, which forms a colored complex that can be measured
Accumulation/utilization of cofactor:
Ex: NADH absorbs strongly at 340 nm, but NAD+ does not, therefore in the reaction of lactate dehydrogenase, the increase of NADH concentration is proportional to the absorbance increase
Coupled reactions: When neither substrate/product or cofactor can be detected easily, it is helpful to observe the reaction if it is coupled
Ex: The formation of l-glutamate is not easily detectable, but it can be coupled with a second reaction that forms NADH, which absorbs at 340 nm
How is KM an approximation for the enzymes affinity to its substrate and an approximation for the dissociation constant of the enzyme-substrate complex?
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The higher KM, the more concentration needed to reach half the velocity, meaning that the affinity of an enzyme for its substrate is weak, and at a small KM, the amount of substrate concentration needed to reach half the velocity is small, meaning that the enzyme has a strong affinity for the enzyme, and has tight binding. Tight binding means that the dissociation constant is small and weak affinity means the dissociation constant is high
What can be used to measure how well an enzyme works?
When you want to compare the efficiency of different enzymes with different affinities for their substrates, you may consider the specificity constant (kcat/KM), which normalizes kcat by the Michaelis constant (Km).
The specificity constant takes both the enzyme’s turnover number and its affinity for the substrate into account, providing a more comprehensive measure of catalytic efficiency for comparing enzymes under different conditions. A perfect enzyme has a very high kcat and very low kM
What are enzyme inhibitors and their categories?
Enzyme inhibitors slow enzyme activity, and two main types of inhibition exist
Reversible enzyme inhibition: Enzyme activity can be recovered by removing the inhibitor via dialysis, gel filtration etc. Reversible
inhibition is characterized by rapid dissociation of the enzyme–inhibitor comp
There are three common types of reversible inhibition, competitive inhibition, uncompetitive inhibition and noncompetitive inhibition
Irreversible enzyme inhibition: Inhibitor binds covalently to the enzyme, which is then irreversibly activated
The inhibition (inactivation) of an enzyme can tell us a lot about the way it works . Enzyme inhibitors are frequently used to define biological phenomena, and are also sought after by big Pharma companies to block enzymes involved in diseases
What are the three types of reversible enzyme inhibition and how do they affect the values of lineweaver-burk plots?
Competitive inhibition involves a inhibitory molecule that binds to the active site of the enzyme, competing with the substrate due to structural similarities. It can be outcompeted with excess substrate, so Vmax is unaffected (y-axis is the same) but because more substrate is needed to compete, Km is increased significantly, so the slope is increased
In uncompetitive inhibition, an inhibitory molecule binds to the ES site, preventing the complex from forming product. This cannot be outcompete like competitive inhibition. Vmax is reduced because there are less productive ES complexes, but Km is actually reduced because the inhibitor binds to ES to form ESI, depleting ES. To maintain the equilibrium between E and ES, more S binds to E, increasing the apparent value of K1 and thereby reducing the apparent value of KM. The slope remains the same, but the y-intercept is now increased because 1/Vmax is smaller. The x-intercept is also more negative.
In noncompetitive inhibition, the inhibitory molecule binds to a site on the molecule other than the active site. The presence of the inhibitory molecule does not affect the binding of the substrate and vice versa. Km is not affected because the binding of the substrate is independent of the inhibitory molecule, but Vmax is reduced because the enzyme-substrate complex does not produce as much product.
What are the 3 types of enzyme catalysis
There are three main types of catalysis
General acid catalysis: Involves the transfer of protons between side chains of the enzyme and the substrate. The most common form of enzyme catalysis
Metal ion catalysis: some enzymes use metal ions (cofactors) for catalysis. The metal ion can act in various ways
Stabilizes transition state
Helps orientate the substrate vs enzyme
Participates in the transfer of electrons/protons between enzyme and substrate
Covalent catalysis: Involves the formation of a transient (temporary) covalent bond between the enzyme and substrate. The covalent bond activates the substrate for further reaction
Name the mechanisms of enzyme regulation discussed in class
Allostery: Inhibition by product or activation by substrate/cofactor
Binding of regulatory subunits
Covalent modification
Degradation of the enzyme
Limited proteolysis
Describe allostery and give an example
Allostery means another site. These are fitting names for allosteric enzymes, which have another binding site other than the active site. An effector molecule binds to a separate site on the enzyme
This is widely used by metabolic enzymes. Often, this occurs through feedback inhibition, where the product of a metabolic pathway will inhibit the enzyme, regulating its activity
Activation by a product generated early on in the pathway can also occur
Allostery is based on the principle of cooperativity. The binding of a small molecule to the enzyme modifies the 3-D structure of the protein and alters its ability to catalyze the reaction
An example of allostery is the enzyme aspartate transcarbamoylase (ATCase)
Involved in the first of a series of reaction leading to the production of cytidine triphosphate (CTP)
CTP then inhibits ATCase by allostery, feedback inhibition
ATP activates ATCase, also by allostery (competes with CTP for binding regulatory sites on ATCase)
