Lesson 3: Enzyme Kinetics Corresponding Kaplan
Describe column chromatography:
Column is filled with polar silica or alumina beads as the stationary phase. The less polar the solvent, the faster it moves.
Describe electrophoresis:
Compounds are subject to an electric filed and separated by mass and charge.
How does comeptitive inhibitor affect vmax and Km?
Does not change Vmax and increases Km
Describe the effects of temperature on enzyme catalyzed reactions?
Enzyme catalyzed reactions tend to double in velocity for every 10 degree Celsius increase in temperature until optimum temperature is reached. If temperature continues to increase past optimum temp, then the enzyme will start to denature and activity will decrease
Describe how molecules can move through the gel medium fast or slow for gel electrophoresis?
Fast: If molecule is small, highly charged, or the elctric field is large Slow: If molecule is large, electrically neutral or placed in a small electric field
Feedback inhibition otherwise known as negative feedback is a type of feedback regulation that is very important. Describe it and why is it important?
Feedback inhibition is where a product further down in the pathway inhibits and enzyme earlier on in the pathway. This is important for homeostasis because once we have enough of a given product we want to turn off the pathway that creates that product rather than creating more.
Describe Hill coefficient and cooperativity:
Hill coefficient: >1 : positive cooperative binding occurs such that as one ligand binds, the affinity for further ligand increases <1: negative cooperativity such that after one ligand is bound, the affinity of the enzyme for further ligand decreases =1: enzyme does not exhibit cooperative binding
Describe chromatography
Homogenized proteins are inserted into a stationary phase and the affinity of that protein for he stationary phase decides how fast or slow a protein elutes. More affinity for stationary phase, slower it elutes. And vice versa
Describe enzyme saturation and how we reach Vmax based on M-M kinetics:
If we have a fixed enzyme concentration, the more substrate we add, the faster the rate of reaction (v) will be. However, there comes a certain point when all enzymes are saturated ( meaning their active sites filled) , and increasing substrate concentration will no longer increase the rate of the reaction. At this point the enzyme is working at max velocity and it is known as vmax
What is the only way to increase vmax?
Increase the enzyme concentration. In the cell this can be accomplished by inducing the expression of the gene encoding the enzyme
Describe uncompetitive inhibition
Inhibitor binds to ES complex preventing release of the substrate
Describe mixed inhibition:
Inhibitor binds to both free enzyme and ES complex but with different affinities unlike non competitive.
Describe salinity and enzyme activity:
It can disrupt Hydrogen bonding, ionic bonding, and cause partial change in conformation of the enzyme and in some cases denaturation
How does noncompetitive inhibition affect Vmax and Km?
It decreases Vmax and does not alter Km
Describe what a cooperative enzyme looks like?
It has multiple subunits and multiple active sites
What does Km tell us?
It helps us determine affinity of the enzyme for its substrate. The higher the Km, the lower the affinity The lower the Km, the higher the affinity
Describe glycosylation as a form of covalent modification of enzymes?
It is the covalent attachment of sugar moeities. Ot can tag an enzyme for transport within the cell, or can modify protein activity or selectivity.
What is Km?
It is the substrate concentration when enzyme is working at half its maximum velocity
What is one risk of using UV spectroscopy to find protein concentration?
It is very sensitive to sample contaminants
What is competitive inhibition?
It is where an inhibitor binds to the active site preventing the substrate from binding there
What does the variable Vmax represent?
Maximum enzyme velocity measured in moles of enzyme per second
Describe bradford assay:
Mixes protein in solution with coomasie blue. Intially, the dye is protonated and green-brown in color. Once in the presence of proteins, it gives up protons upon binding to amino acids turning blue in the process. Ionic interactions between the dye and the amino acids stabilizes the blue form of the dye. Using a standard curve base don known concentration of protein to absorbance of the coomasie blue, we can then figure out concentration of the unknown protein sample. This method is more accurate in the presence of one type of protein and less accurate in the presence of multiple types.
What two methods can be used to determine protein structure?
NMR and Xray crystallographt
In electrophoresis, how do negatively charged compounds and positively charged compounds work?
Negatively charged compounds will move towards the positive charged anode Positively charged compounds will move towards the negatively charged cathode
Does increasing substrate concentration overcome non competitive inhibition?
No
Describe covalent modification of enzymes (phosphorylation, dephosphorylation):
Phosphorylation and dephosphorylation is the addition and removal of a phosphate group from an enzyme.We cannot be certain whether removal or addition causes activation unless we test it experimentally.
What is the standard gel used for protein electrophoresis?
Polyacrylamide
Describe isoelectric focusing
Proteins are placed in a gel that has a pH gradient ( acidic at the positive anode and basic at the negative cathode), and are separated on the basis of their isoelectric point. Positively charged proteins will move towards the cathode and negatively charged proteins will be moving towards the anode. WHen they reach a pH that is equal to the pI, then the protein becomes neutral and stops moving.
What is irreversible inhibition?
Refers to the prolonged or permanent inactivation of an enzyme, such that it cannot be easily renatured to gain function
Describe size exclusion chromatography
Stationary phase beads contain tiny pores that allow only certain size molecules through. Typically, the larger the molecule, the faster it will elute in this column.
Describe affinity chromatography
Stationary phase is typically beads that have receptors for a specific protein or antibody to the protein. Once desired protein is tuck in column, free receptors are added to outcompete bead receptors for the protein.
Describe R state and T state for cooperative enzymes?
Subunits and enzyme may exist in either the high affinity R (relaxed) state or low affinity T (tense) state. Binding of a substrate encourages transition of other subunits from T state to R state which increases likelihood of substrate binding to the other subunits. Loss of substrate encourages transition from R state to T state and promote dissociation of substrates from the remaining subunits.
What is retention time in relation to chromatography?
The amount of time a compound spends in the stationary phase
Describe SDS Page:
The detergent SDS disrupts all noncovalent interacts denaturing the protein. It also gives the protein a negative charge. As proteins move through the gel, the only variables affecting velocity is E ( strength of the electric field), and f (frictional coefficient which depends on mass). This means that proteins can only be separated by mass
When an enzyme shows a sigmoidal curve instead of a hyperbolic curve on the M-M plot, what does that mean?
The enzyme exhibits cooperativity.
Can proteins be recovered after native page?
The native protein can be recovered but only if the gel has not been stained because certain stains denature proteins
What are zygmogens?
They contain a catalytic domain and regulatory domain. The regulatory domain must be either removed (cleaved) or altered to expose the active site
Describe Native PAGE:
This is a method for analyzing proteins in their native state. This separates proteins by both mass and charge through mass to charge ratios. Since may proteins may move the same distance due to mass to charge ratios, this is mainly useful for proteins that are similar in wither mass or charge, and then watching how they separate based on the other variable.
How do you determine the amino acid composition of small proteins?
Through edman degradation where proteins up to 50-70 amino acids sequentially has their N terminal amino acid removed that can then be analyzed by mass spectroscopy
What methods can be used to determine protein concentration?
UV spectroscopy and bradford protein assay with the help of Beers Lae (A=Elc)
How do you determine the amino acid composition of larger proteins?
Use specific proteolytic enzymes such as trypsin, to cleave protein into smaller fragments. From there you can then use edman degradation to find the composition of the amino acids in the smaller fragments
How does uncompetitive inhibiton alter Vmax and Km?
Vmax and Km are proportionally lowered
Describe how Km and Vmax are altered for mixed inhibitor:
Vmax is lowered Km is increased if it binds to free enzyme Km is decreased if it binds to ES complex
What is Kcat?
Vmax/[Et] It is also known as turnover number and it measures the number of substrate molecules converted to product per enzyme molecule per second
What is feed forward regulation?
When enzymes are regulated by intermediates that precede the enzyme in the pathway
What is feedback regulation?
When enzymes are regulated by products further down a given metabolic pathway
Is it possible for enzymes that were denatured at high temps to regain function if cooled?
Yes it is possible but only for a few enzymes
Are cooperative enzymes subject to activation or inhibiton?
Yes they are. Both competitively and through allosteric sites
Describe pH and enzyme activity:
pH not only affects ionization of the active site, but it also can lead to denaturation of the enzyme Any deviations from the optimum pH lead to denaturation and decreased activity of the enzyme
Michaelis-Menten equation
v = (vmax [S])/(Km + [S])
How do we calculate migrational velocity in electrophoresis and what does this equation mean?
v= Ez/f Velocity is directly proportional to electric field strength and net charge Velocity is inversely proportional to frictional coefficient which depends on mass and shape of molecule
For the lineweaver burke plot, what represents this: x intercept: y intercept: Slope:
x intercept: -1/Km y intercept: 1/Vmax Slope: km/vmax
What is the equation used to come up with the line weaver burke plot that is equivalent to the michaelis menten plot?
1/v = (km/vmax) (1/s) + 1/vmax
What is the catalytic efficiency of an enzyme?
A second order reaction dependent of both enzyme and substrate concentration Kcat/Km Higher the kcat and lower the Km the better catalytic efficiency
For competitive inhibition, what can be done to overcome the inhibition?
Adding more substrate because if there is more substrate present than inhibitor, the inhibitor is more likely to bind to it than inhbitor
Describe allsoteric inhibitors and activators for allosteric enzymes?
Allosteric enzymes contain another site apart from the active site which is called the allosteric site and it is here where allosteric inhibitors and activators can bind. Allosteric activators will result in a shift that will make the active site more available for binding substrate, whereas inhibitor will make it less available.
Describe X-ray crystallography
An Xray diffraction pattern is generated and the the small dots in the diffraction pattern can then be interpreted to determine protein's structure
Describe ion exchange chromatography:
Beads in column as stationary phase are coated with charged substances so they attract or bind substances with opposite charge. If a compound of similar charge is placed into column, it will elute faster than a compound of opposite charge. After all other compounds have eluted through this column, a salt gradient is used to elute the charged molecules that got stuck in the column
Describe noncompetitive inhibition
Binding of inhibitor to an allosteric site. In non competitive inhibition, the binding of inhibitor to free enzyme and enzyme substrate complex is equally done
How does one determine protein activity?
By monitoring a known reaction with a given concentration of substrate and comparing it to a standard. Reactions with color changes become very applicable for this
