Lectures 8-19 Flashcards
What is the delta G of a thermodynamically favored event?
Negative deltaG. The G (free energy) of the products is lower than that of the reactants.
Why does a thermodynamically favored process not necessarily proceed spontaneously then?
Often there is a high-energy intermediate or transition state.
What is the energy required to reach the transition state?
The activation energy.
Enzymes do what?
They form transitory bonds with the reactants. These enzyme-reactant complexes have relatively low energy and make the reaction a more likely even by lowering the activation energy.
Do enzymes affect the equilibrium of the reaction?
NO. Equilibrium depends solely on deltaG. Spontaneously, given enough time, product would accumulate, and the reverse reaction to form the initial reactants would be even less likely. At steady state, the relative concentrations of reactants and products would be the same, whether the reaction were catalyzed or not. The enzyme changes ONLY the reaction rate.
What is Le Chatelier’s principle?
A change in the concentration of reactant or product will shift the equilibrium of the reactions involved. AKA if you have a reaction A->B->C , Continual depletion of B in the reaction catalyzed by Enzyme 2 increases the rate of A->B.
Describe the Lock and Key method.
The lock-and-key model postulates that the binding site is a static, rigid structure that selects for substrates that fit optimally (not only with respect to shape, but also in the potential to form bonds with residues in the active site). This model has largely been superseded by the induced-fit model, which offers a better account of the thermodynamic basis for catalysis.
Describe induced fit.
In the induced-fit model, the active site of the enzyme in its unbound state includes a low-affinity binding site for the substrate. When the substrate binds to this site, the enzyme undergoes a conformational change (see figure below) that increases the active site’s affinity for the substrate, but which requires that the substrate undergo steric changes that bring it closer to a transition state.
Use metal rod binding to explain the advantage of induced fit over lock and key.
1st screenshot.
When is a reaction first order?
The initial rate is directly proportional to [S]. Rate here is dependent on [S].
When is a reaction zero order?
At very high substrate concentrations, the relationship flattens, and V0 becomes constant. In this region, where rate is independent of [S], the reaction is said to be zero-order.
What is Vmax?
The highest possible reaction rate for a given catalyzed reaction.
How do you determine which enzyme out of two has a higher affinity for the substrate?
Whichever enzyme reaches vmax at a lower substrate concentration.
What is Km
The substrate concentration at which V0 is 50% of Vmax
What is the Michaelis-Menten equation?
When [S] is much lower than Km what can we do?
We can ignore [S] in the denominator, and since Vmax and Km are constants, V0 is directly proportional to [S] (first-order behavior).
When [S] is much larger than Km what can we do?
When [S] is far larger than Km, we can ignore Km in the denominator, and V0 = Vmax (zero-order behavior).
Rearrange the Michaelis Menten equation.
1/Vo=
How does increasing the concentration of the substrate affect a competitive inhibitor?
increasing the concentration of the substrate can displace a competitive inhibitor. At sufficiently high substrate concentrations, which effectively deny the inhibitor access to the binding site (all of the binding sites are occupied by S), the enzyme catalyzes the reaction at Vmax.
How is the Y-intercept on a Lineweaver-Burk plot (1/Vmax) affected by a competitive inhibitor?
It is not affected, it remains the same.
What value changes with a competitive inhibitor?
The apparent Km because it takes more substrate (or higher [S]) to displace the inhibitor and occupy the number of binding sites that yield 50% of Vmax. As a result of this the Lineweaver-Burk plot (Km/Vmax) becomes STEEPER and the X intercept (-1/Km) shifts to the RIGHT. See second screenshot for plot.
What is the equation used to describe the relationship of Vo and [S] in the presence of a competitive inhibitor?
How does increasing the substrate concentration affect a non-competitive inhibitor?
It doesn’t. Even at saturating substrate concentrations, where all of the enzyme is substrate-bound, Vmax is reduced from its value in the absence of inhibitor.
How is the Lineweaver-Burk plot altered for a non-competitive inhibitor?
Since Vmax is reduced, the y-intercept (1/Vmax) increases. Because substrate binding is unaffected, Km stays the same and therefore so does the X-intercept (-1/Km). This causes the slope of the line to become steeper or INCREASE. See third screenshot.
What is uncompetitive inhibition?
Uncompetitive inhibitors can only bind the enzyme after substrate has bound, since formation of the ES complex creates the site for the inhibitor.
How is the Lineweaver-Burk plot affected by uncompetitive inhibitors?
When the inhibitor binds, it stabilizes the ES complex, thus slowing the dissociation of the substrate from the enzyme
(ESE +S), and this reduces Km, shifting the x-intercept to the left. The inhibitor also interferes with the catalytic activity of the enzyme, so Vmax decreases as well. In the presence of an uncompetitive inhibitor, the Lineweaver-Burk plot is shifted UPWARD and PARALLEL to the no-inhibitor setting, since Km and Vmax decrease to similar degrees.
When a regulatory enzyme is regulated by the binding of a modulator this is called…
An allosteric enzyme.
What can modulate an allosteric enzyme?
Either the substrate itself (in which case the modulatory site is the same as the active site) or some other molecule that acts outside the active site.
What is homotropic modulation?
When the substate itself is the modulator of the enzyme. Such enzymes have multiple active sites, usually on different subunits, and the binding of the substrate to the first active site increases or decreases the affinity of other active sites for the substrate.
What is positive cooperativity?
When the binding of a substrate increases the affinity for more binding to the enzyme (O2 on hemoglobin).
What curve demonstrates positive cooperativity?
Sigmoidal instead of hyperbolic.
What is heterotropic cooperativity?
When the enzyme responds to a modulator other than its substrate. Often the product. This is negative feedback.
What is an example of negative feedback where the product is many steps beyond that?
Heme is the product and feeds all the way back to give feedback to gamma-aminolevulinic acid synthetase which catalyzes the rate-limiting step of the reaction 8 steps earlier.
Positive modulators affect Km and Vmax how?
Usually decrease Km with no effect on Vmax.
Negative modulators affect Km and Vmax how?
negative modulators can produce an apparently competitive inhibition (increased Km, no effect on Vmax; see middle panel, lower plot), or apparently non-competitive inhibition in which Vmax is reduced but Km unaffected (right panel).
Describe Thr286, how it gets activated and what happens when it does.
Ca2+ is a ubiquitous second messenger whose effects on proteins often depend on its complexing with calmodulin, a small, highly conserved protein that can bind as many as four Ca2+ ions. These Ca2+ ions induce a conformational change in calmodulin, exposing a hydrophobic surface that can bind to hydrophobic pockets in other proteins and acting as a modulator (usually positive).
One of the proteins to which Ca2+/calmodulin binds is CaMKII (see figure on next page), a serine/threonine protein kinase (that is, it phosphorylates those residues, provided that they are in a particular context with respect to neighboring amino acids and geometry). CaMKII, which is one of the most abundant proteins in the brain and implicated in memory formation, is comprised of 12 homologous subunits (it is a dodecamer). Each of the 12 subunits is a single polypeptide that contains a catalytic domain, a regulatory domain, a hinge that connects them, a phosphorylatable threonine (Thr286) within the hinge region, and a hydrophobic Ca2+/calmodulin binding pocket. In the inactive resting conformation, the hinge is closed and the regulatory domain makes the catalytic domain inaccessible to potential substrates.
A rise in intracellular Ca2+ produces an increase in the concentration of Ca2+/calmodulin, which binds to its hydrophobic site in CaMKII and causes CaMKII to adopt an open conformation that exposes the catalytic domain as well as Thr286. CaMKII now can phosphorylate its substrates, but the level of Ca2+/calmodulin usually drops quickly, so it quickly dissociates from CaMKII and the enzyme again is inactive. This is how modulators work, in a readily reversible manner. However, if the Ca2+ increase was particularly large, Ca2+/calmodulin levels stay high for longer than usual, and this gives CaMKII subunits the opportunity to phosphorylate the exposed Thr286 of a neighboring subunit. Once the Thr286 is phosphorylated, the hinge cannot close even after Ca2+/calmodulin has dissociated, and CaMKII remains active for much longer – until a protein phosphatase called PP1 can remove the phosphate group from Thr286.
What is a zymogen?
An inactive precursor that has to be cleaved to become an active enzyme.
What is a kinase?
enzymes that transfer a phosphate group from a high-energy donor (such as ATP) to a substrate are called kinases (for example, cAMP-dependent protein kinase)
What is a cofactor?
Many enzymes require an additional small molecule, called a cofactor, to perform their catalytic function. In some cases, the cofactor is covalently bound to the protein part of the enzyme, in which case the protein itself is called the apoenzyme and the full complex is the holoenzyme.
What is Kcat?
The number of operations that a single molecule of enzyme can perform per second. Kcat is referred to as the turnover number.
What is the equation for Kcat?
Kcat= Vmax/[Et]
Where [Et] is the molar quantity of enzyme present in the reaction.
What is the best estimation of the efficiency of an enzyme under physiological conditions?
The specificity constant, which is Kcat/Km. This value (with units M-1s-1) indicates how efficient the enzyme is when free binding sites are abundant.
If Kcat/Km is large what does that indicate?
If kcat/Km is large, then the reaction may proceed at a reasonable rate even if the enzyme is not highly expressed (that is, [ET] is small) or if substrate concentration is low.
What does the Na/K ATPase pump do?
Exports 3 Na+ molecules and imports 2 K+ molecules at the expense of one ATP.
What does the Na/K ATPase pump look like in the E1 conformation?
It has 3 high affinity Na+ binding sites facing the cytosol that become occupied. There are also two low affinity unoccupied K+ binding sites.
How does Na/K ATPase pump change conformation from E1 to E2?
ATP binds to the pump and is hydrolyzed by the pump’s ATPase activity, and a high-energy phosphate bond is formed with an aspartate residue on the cytoplasmic side of the pump.The energy of the phosphate is used to drive a conformational change in the protein to E2, a process called the “power stroke”. As a result, the Na+ ions move to low-affinity sites that are exposed to the extracellular space, where they are weakly bound. The E2 conformation also presents two high-affinity K+ binding sites to the extracellular side
How does Na/K ATPase pump change conformation from E2 to E1?
The three Na+ ions diffuse away from their low-affinity sites and into the extracellular space. At the same time, two K+ ions from the outside bind their high-affinity sites, causing hydrolysis of the aspartyl-phosphate bond. The loss of the phosphate group returns the pump to its E1 state, transferring the K ions to their low-affinity binding sites that face the inside of the cell, from which they dissociate into the cytoplasm.
How is the Ca2+ concentration kept low within the cell?
The SERCA P-class pump, which sequesters Calcium from the cytosol into the sarcoplasmic reticulum. It operates very similarly to the Na/K ATPase pump: when Ca2+ is bound to the high affinity cytosolic sites, ATP is hydrolyzed, the liberated phosphate group forms a high- energy bond with an aspartate on the cytoplasmic side, inducing a conformational change (to E2) that closes off the Ca2+ pocket from the cytoplasmic side, trapping Ca2+ in the protein.
What do P-class pumps move?
Only ions.
What do V/F pumps move?
ONLY protons.
What do V-class pumps do?
V-class pumps contribute to the acidification of organelles such as lysosomes by pumping protons from the cytoplasm to the lumen of the organelle.
What do F-class pumps do?
F-class pumps, one type of which (F0-F1 ATPase) is highly expressed in mitochondria, typically run “in reverse”, using the movement of protons down their gradients to synthesize ATP.
How do ABC pumps bind ATP?
Through conserved regions called ATP-binding cassettes (thus, “ABC”).
What do ABC pumps transport?
Unlike the P- and V/F classes, ABC pumps often transport uncharged and even hydrophobic molecules.
What are Multi-drug resistance (MDR) proteins?
A type of ABC pump. They are highly expressed in epithelial cells. They transport small, polar molecules, including some products of normal metabolism, but they can also pump a wide variety of drugs out of cells. Thus, tumors that overexpress MDR proteins are resistant to treatment by multiple and unrelated anticancer drugs.
What is the cystic fibrosis transmembrane regulator (CFTR)?
An ABC pump expressed in the lung and other organs. Although structurally an ABC-class pump, it has no known “pumping” function. However, it incorporates a channel that is permeable to Cl–, and that is regulated by protein kinase A. Cystic fibrosis has been linked to loss-of-function mutations in CTFR, which reduce Cl– transport across pulmonary epithelial cells. As a result, the mucus secreted by these cells becomes excessively viscous, compromising gas exchange and predisposing the lung to infection.
How do transporters differ from pumps?
Transporters, like pumps, bind solutes and undergo conformational changes to ferry them across the membrane. In contrast to the pumps, the transporters have no ATPase activity; rather, they rely on existing gradients to move solutes.
What does a uniporter do?
They conduct a single species of molecule down its gradient, facilitating a thermodynamically favored process by circumventing the hydrophobic barrier of the lipid bilayer (facilitated diffusion).
What do co-transporters do?
They couple the thermodynamically favorable movement of one type of molecule (down its gradient) to the unfavorable movement of another (secondary active transport).
Symporter
Transporter moving solutes in the same direction across the membrane.
Exchanger
Also called an anti porter is a transporter move two solutes in opposite directions.
Describe the action of a Glucose Unitransporter (GLUT)
GLUTs bind a single molecule of glucose at a time. A conformational change exposes the glucose-binding site alternately to the extracellular and intracellular sides, and the rate of cycling is accelerated by occupation of the binding site in either conformation. In most cells, the concentration of glucose is higher outside the cell than inside, so the extracellular binding site is more likely to become occupied. The intracellular concentration of glucose is kept low by the rapid phosphorylation of glucose to glucose-6-phosphate. Upon the conformational change, the glucose is exposed to the low concentration inside the cell, and it diffuses away from the binding site. GLUT spontaneously returns to its initial conformation, and another molecule of glucose can bind from the outside.
Describe the Na+/Ca2+ exchanger.
Na+/Ca2+ exchanger is an antiporter that couples Na+ entry to Ca2+ efflux. Thus, it serves a role in Ca2+ handling that is complementary to the sequestration performed by the SERCA pump, and in cardiac cells it contributes to Ca2+ clearance more than SERCA does. It has a stoichiometry of 3 Na+ : 1 Ca2+, so it is electrogenic, accumulating positive charges inside the cell.
Which SLGT Is expressed in the early part of the renal tubule?
SLGT2.
What does SLGT2 do?
In the early part of the tubule that is near the glomerulus, the concentration of glucose in the urine is relatively high, so the transporter that is expressed in this region (SGLT-2) doesn’t need to work against a large concentration gradient in taking up glucose into the cell. The stoichiometry of SGLT-2 is 1 Na+ : 1 glucose, which is sufficient to accumulate glucose against a 100-fold gradient.
Which SLGT is expressed later on in the kidney tubule?
SLGT1.
What does SLGT1 do?
There is so little glucose later in the tubule rabsorption becomes more of a challenge since the glucose gradient strongly resists movements from the lumen into the cell. SGLT-2 would be ineffective. Cells in this part of the tubule express the SGLT-1 symporter, whose stoichiometry is 2 Na+ : 1 glucose. By allowing the Na+ gradient to deplete by 2 molecules for each glucose molecule reabsorbed, SGLT-1 can work against a glucose gradient up to 30,000-fold. As a result, essentially no glucose is excreted in the urine.
Which channels provide predominant membrane conductance?
Channels that are selective for K+ usually provide the predominant membrane conductance.
What is equilibrium potential? (Veq)
Also known as reversal potential (Vrev), it is the value of Vm when the concentration gradient and the electrostatic force are equal and opposite.
Nernst equation:
Veq(S)=RT/ZsF*ln([So]/[Si])
Simplified…
Veq(S)=58/Zs*log([So]/[Si])
If there is 150 K+ inside the cell and 4K+ outside the cell calculate Vk.
Vk=58/1*log(4/150)= -91mV.
At resting -71 membrane potential if you allowed the flow of K+ ions what would happen in which direction?
If the resting membrane potential is –70 mV, the opening of K+ channels will cause the cell to hyperpolarize. The net movement of K+ in this case (the K+ current) is toward the outside of the membrane. The movement of cations out of the cell is referred to as an outward current, and outward currents hyperpolarize membranes.
Which kind of current depolarizes membranes?
Inward current, or flow of positive ions into the cell.
What is the driving force?
Driving force= Vm- Veq
So if membrane is -70 and Vk is -91, the driving force is +21 mV.
What is conductance?
The ability of a material to carry current.
