biochem exam 4 (transporters and signaling)

what does a transporter protein do?

-a transporter protein reduces the energy barrier required to diffuse a polar molecule through a membrane

-favorable interactions between the transporter protein and substrate

what does the GLUT1 transporter do?

-GLUT1 is a passive (going don electrochemical gradient) transporter of glucose

-glucose is polar, is has 6 OH

how does the passive glucose transporter work?

-glucose from the blood plasma binds to T1

-glucose binds → lowers activation energy for conformational change from T1 (facing outside) to T2 (facing inside)

-now glucose can be released from T2 into the cytoplasm

-glucose has been unbound and the transporter returns to T1

what is ligand gated vs voltage gated?

-ligand gated: the ligand is what causes the signal like glucose with GLUT1

-voltage gated: a certain amount of electrochemical potential enables channel

• important in neurons where rapid changes in activity of ion channels causes changes in membrane potential/voltage

• voltage= unequal [ion] on either side of membrane

how does the ion-selective channel, the potassium channel work?

-cone of alpha helices

1. hydrated potassium enters the chamber

2. the channel narrows and potassium dehydrates and interacts with the helix carbonyls

3. carbonyls go down in energetic stabilization and potassium rehydrates

what would happen if sodium entered the potassium channel?

-if sodium enters, the channel collapses

-potassium and sodium are different sizes, sodium is too small and the carbonyls are too far apart to coordinate with sodium

-the active sites are perfectly sizes for a specific ion radius

what is specificity of signal transduction?

-specificity: receptor protein as a binding site is complementary to a specific signal molecule

what is modularity of signal transduction?

-modularity: proteins with multivalent affinities can mix and match to form enzyme complexes

-typically regulated by phosphorylation

what is amplification of signal transduction?

-amplification: activated enzyme can activate other enzymes in an “enzyme cascade”

what is desensitization/adaptation of signal transduction?

-desensitization: if a signal is continuously present, activation of the receptor triggers a negative feedback loop to shut off the receptor

what is integration of signal transduction?

-multiple signals can regulate a single response

-observed response is a combination (integration) of each pathway

what is localization of signal transduction?

-if enzymes that destroy intracellular messages are near receptors, they prevent signals from diffusely propagating outside of a local region

-don’t want your signal going everywhere

what are G-protein coupled receptors (GPCRs)?

-plasma membrane receptors

-binding of a ligand activates the receptor

how do GPCRs work and what is an effector enzyme?

-G protein exchanges GDP for GTP

-part of G protein dissociates + binds effector enzymes → regulates activity and produces signal

-effector enzyme alters concentration of metabolites to effect downstream activities

what is a primary effector and what is a secondary effector?

-the ligand is the primary effector (initial signal)

-the activated G protein activates the effector which produces the secondary effector

adrenergic receptors are GPCRs, what is the agonist and the antagonist?

-agonist: binds GPCRs and activates, can be natural ligand or structural analog

-antagonist: binds GPCRs and prevents activation by blocking agonists from binding (kind of like competitive inhibitors)

what is the primary effector of the B-adrenergic receptor?

-epinephrine which binds the B adrenergic receptor

what is associated with the B adrenergic receptor? what are its features?

-a G protein

-alpha + beta: a part of gamma, lipid anchors that anchor it to the membrane

-gamma: the big block, attached to the edge of the plasma membrane

what is bound to the alpha when inactive? how does this change when epinephrine binds?

-GDP bound to alpha when inactive

-upon association of epinephrine, change in intracellular domain → GDP dissociates and GTP binds alpha

now alpha is activated, what happens next?

-alpha + GTP travels across the membrane to find the effector: adenyl cyclase effector protein

-now adenyl cyclase effector protein catalyzes the reaction of ATP → cAMP

what happens upon the conversion of ATP → cAMP?

-cAMP interacts with PKA which then activates other proteins, leading to a signal cascade

what is the structure of PKA? what happens to it when cAMP binds?

-PKA has 2 catalytic subunits and 2 regulatory subunits

-catalytic subunits are inactivated by regulatory subunits

-cAMP binding to regulatory subunits releases catalytic subunits which are now active

what is the goal of signal amplification of the B adrenergic receptor?

-the goal is fight or flight, and getting cells energy from glycogen stores

why does the signal need to be terminated?

-we don’t want to deplete the glycogen sugar stores by regenerating those initial constructs

-few things are constitutively expressed

how is the signal terminated?

-the epinephrine gets degraded and its concentration of epinephrine in the blood drops below Kd (binding affinity) for the receptor

-you want to regulate the signal termination from the top, the initial primary effector so as not to be wasteful

what happens when epinephrine dissociates from the GPCR?

-GTP bounds to alpha is converted to GDP by intrinsic GTPase activity

-this intrinsic GTPase activity is slow (kind of moderating the time window of the signal)

-GTPase activator proteins (GAPs) stimulate GTPase activity to speed it up

even if you turn the G protein receptor off, you still have some cAMP left that could amplify signals. how do you terminate that?

-cAMP needs to be hydrolyzed to AMP by cyclic nucleotide phosphodiesterase

ok, you’ve gotten rid of the GTP and the cAMP but proteins are still phosphorylated from when PKA was working. what do you do about that?

-phosphoprotein phosphatases dephosphorylate proteins that were phosphorylated by PKA

how does the G protein know if GTP is there or not?

-the difference between GTP and GDP is just a phosphate

-the G protein has floppy arms (switch 1 and switch 2) that sense the 3rd phosphate of GTP

-the furthest negatively charged phosphate of GTP (the gamma phosphate) is salt bridging with glycine/threonine of the switches, they are exposed when GTP is bound

-when GDP is bound the switches move inward and are not exposed

desensitization is different from the signal ending. how does it work?

1. binding of epinephrine → activated GPCR. G alpha activates another protein and G beta and gamma are left behind

2. G beta and gamma recruit BARK to membrane to phosphorylate Ser at the C term of receptor

3. B arrestin binds phosphorylated C term domain of receptor

4. receptor-arrestin complex enters cell by endocytosis

5. in the endocytic vesicle arrestin dissociates and the receptor is dephosphorylated and returned to cell surface

what kind of amino acid do you need to phosphorylate?

-you need a nucleophile to phosphorylate

what is metabolism?

-metabolism: the sum of all chemical transformations in a cell/organism

metabolism is driven by a series of enzyme catalyzed reactions. what is anabolism/catabolism?

-anabolism: biosynthesis that requires energy

-catabolism: degradative steps of metabolism that produces energy

why do cells require a source of free energy?

-cells operate under constant pressure and temperature

-this means heat transfer cannot be a source of energy, where would the heat go if everything is the same temp

where does free energy come from?

-free energy (G) is the amount of E to do work

-this energy comes from food (chemicals) and the sun (photons)

what is delta G knot vs. delta G’ knot?

-delta G knot: standard pressure, temperature, concentration

-delta G’ knot: standard free energy, under cellular conditions

what is the significance of Q?

-changing the concentration of metabolites will change free energy

-this is what Q represents, the measure of reactants/products at a given moment in time

what is the reaction when Q=1, Q<K, Q>K?

-when Q=1 the reaction is in standard state

-when delta G=0 the reaction is at equilibrium, Q=K

-when Q<K then delta G is negative because less product/more reactant and the reaction proceeds to the right

-when Q>K then delta G is positive because more product/less reactant and the reaction proceeds to the left

how is PKA activated by cAMP?

-PKA is allosterically activated by cAMP