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Consider the following pathway called A with 5 molecules numbered 1-5 A: 1--> 2--> 3--> 4--> 5 if the reaction of the rate determining step is decreased, how will the speed with which substance 5 is made change?

A reaction can not occur faster than its slowest step. Therefore, the rate determining step effectively limits the overall rate of a reaction. If you decrease the rate determining step, then you are decreasing the overall rate of the reaction making it slower.

How are PFK1 and FBPase 1 reciprocally regulated?

AMP reciprocally regulates both enzymes in the reverse pathway AMP increases PFK1 affinity and decreases FBPase1 affinity ATP is high - ATP status is not enough information to make glucose --> just ate - don't need to keep making glucose ADP is a sign of ATP hydrolysis - energy status is low why AMP? adenylate kinase - recovers ATP from ADP 2ADP --> AMP + ATP

How is pyruvate kinase regulated allosterically?

ATP - slows it down - energy status F16BP- feedforward activation - prevents bottle neck of metabolites acetyl CoA and Fatty acids - slow it down

What happens to ATP synthesis in the presence of an uncoupled? why?

ATP synthesis is stopped in the presence of uncoupler but oxygen consumption continues so electron flow continues uncoupler undoes proton gradient - alternate path between space and matrix for protons ATP synthesis is dependent on proton movement no H+ movement then no ATP synthesis

The allosteric binding site for acetyl CoA on pyruvate carboxylase is mutated, decreasing affinity for acetyl CoA. What would happen in gluconeogenesis?

Acetyl CoA is an activator of pyruvate carboxylase gluconeogenic flux is lowered

ATP hydrolysis is much more favorable in cells. The concentration in an average cell are: ATP 5 mM, ADP .5mM, and Pi 1mM. What is the actual value of ATP in an average cell at 37 degrees C? standard free energy change = 30.5 kJ/mol

Actual Free energy change= standard free energy change + RT(products/reactants) Actual Free energy change= (-30,500 J/mol) + (8.314 J/molK)(310K)ln((5x10^-4 M)(1x10^-3M)/5x10^-3 M) = -54,238 J/mol

How are actual and standard free energy related ?

Actual Free energy change= standard free energy change + RTln(products/reactants)

What creates covalent and allosteric forms of regulation?

Allosteric--> encouraged by concentration of substrate, ability to noncovalently bond, presence of inhibitors Covalent--> ability to covalently bond

The reaction catalyzed by pyruvate carboxylase is an example of an anaplerotic reaction. What does this mean? Why does it make sense that acetyl CoA activates this reaction?

Anaplerotic reactions replenish depleted cycle intermediates when acetyl CoA builds up, flux of TCA is slowed to correct for low flux, it makes sense for the acetyl CoA to convert to pyruvate to oxaloacetate why do you need to replenish? because they are amphibolic

One of these two regulatory proteins is stimulated by insulin. This same regulatory protein is also regulated allosterically by calcium. Which of the two proteins is regulated this way, how do both of these mechanisms of regulation make sense?

Calcium regulation of a phosphatase Calcium is high in muscle contraction --> muscle needs ATP--> PDH needs to be active insulin regulation of phosphatase --> blood sugar increases --> increase activity of PDH --> to create FA need to go through acetyl CoA

Why do patients with hereditary acyltransferase deficiency have muscle weakness? Why is it much more severe during fasting?

Carnitine acyltransferase II releases acetyl CoA in mitochondria if it is deficient, then there is reduced acetyl CoA which affects TCA cycle this reduces ATP production and muscles require ATP lose beta oxidation if carnitine acyltransferase II is deficient, robbed muscle of fuel source at rest, fast --> low glucose levels --> muscles makes it drop even more

Most of our carnitine comes from consumption of red meat. Why might you add a carnitine supplement to your diet?

Carnitine helps you transport FA into mitochondria if you don't transport FA into mitochondria then you could die

What are the five coenzymes, which are required in catalytic or stoichiometric amounts.

Catalytic: TPP- steps 1 and 2 lipoic acid- steps 2,3,4 FAD- steps 4 and 5 Stoichiometric: CoA- step 3 NAD+ - step 5

What are chylomicrons?

Chylomicrons are tiny fatty droplets composed of triglycerides, small amounts of phospholipids, cholesterol, free fatty acids, and some protein. a lipoprotein packaged triacylglycerol with proteins in blood

How does citrate affect flux through glycolysis and gluconeogenesis?

Citrate activates FBPase1 and inactivates PFK1 CAC flux is low if citrate builds up--> energy status must be high glycolysis --> fast --> insulin is present PDH would be inhibited by ATP but insulin overrides this build up of G6P if inhibit PFK1 get NADPH to power FA synthesis

Perhaps the biggest regulator of flux through the CAC cycle is availability of acetyl CoA and oxaloacetate. What enzyme would you expect to be regulated by these molecules?

Citrate synthase through substrate availability oxaloacetate availability --> what is always limiting glycolysis --> high in oxaloacetate and acetyl CoA gluconeogenesis--> high in acetyl CoA

The uncoupler 2,4 - dinitrophenol (DNP) was once used as a weight loss drug. Why would an uncoupler cause weight loss?

DNP creates an alternate route for protons to go back to matrix down concentration gradient. Energy from proton flow is released from heat obesity decrease of brown adipose not making much ATP --> only glycolysis + little for CAC proton gradient never builds up so NADH never builds up no signs to regulate energy production --> burn everything I got as fast as I can

Which steps are points of regulation in glycolysis? why?

Don't want to waste ATP Steps 1 (hexokinase), 3 (PFK), and 10 (Pyruvate kinase)

Under normal conditions, the activity of E1 is 50pmol of substrate 2 produced per 10^6 cells per second and that of E2 is 40pmol of substrate produced per 10^6 cells per second. what is the direction and rate of metabolic flux when an inhibitor reduces activity of E1 by 10%?

E1: 50 pmol--> 45 pmol 45-40 = 5 pmol per 10^6 cells per second of substrate 3 is produced forward direction

Under normal conditions, the activity of E1 is 50pmol of substrate 2 produced per 10^6 cells per second and that of E2 is 40pmol of substrate produced per 10^6 cells per second. what is the direction and rate of metabolic flux when an activator increases the activity of E2 by 10%?

E2: 40 pmol--> 44 pmol 50-44 = 6 pmol per 10^6 cells per second of substrate 3 is produced forward direction

Under normal conditions, the activity of E1 is 50pmol of substrate 2 produced per 10^6 cells per second and that of E2 is 40pmol of substrate produced per 10^6 cells per second. what is the direction and rate of metabolic flux when E2 activity doubles?

E2: 40 pmol--> 80 pmol 50-80 = -30 30 pmol per 10^6 cells per second of substrate 2 is produced backwards direction

what would happen to the ETC if one could artificially maintain the [H+] in the intermembrane space at very high levels?

ETC would stop working The ETC normally pumps H+ out of the matrix into the intermembrane space, but if the intermembrane space has a high concentration of H+ already, then the protons don't want to leave the matrix. If H+ doesnt diffuse to the intermembrane space, then it si not coupled to drive ATP synthesis

Atractyloside inhibits ATP/ADP translocase, the transporter that move a molecule of ATP from the matrix to cytosol in exchange for an ADP moved into the matrix. What would happen to the carriers of the ETC, to the proton gradient, to ATP synthesis, and to TCA flux?

Energy status in matrix is high --> decrease ATP synthesis --> proton gradient builds up flux through TCA stops because you don't have NAD+ carriers are reduced

What effect would epinephrine have in muscle? glucagon?

Epinephrine--> breakdown of glycogen --> Gs--> adenyl cyclase --> cAMP --> activates PKA Muscles don't have glucagon receptors --> muscle has no use for glucagon because glycogen is consumed by the muscle and not released back into the bloodstream

Consider the following pathway called A with 5 molecules numbered 1-5 A: 1--> 2--> 3--> 4--> 5 If the step from 2 to 3 is the rate determining step, what intermediates will accumulate?

Everything behind the rate determining step will accumulate 1 and 2 will accumulate

Consider the following pathway called A with 5 molecules numbered 1-5 A: 1--> 2--> 3--> 4--> 5 What intermediates would accumulate if the step from 4 to 5 was the rate determining step?

Everything behind the rate determining step will accumulate 1, 2, 3, 4

How does F2,6BP alter the activities of PFK1 and FBPase1?

F26BP is an allosteric effector for PFK1 and FBPase1 F26BP binds to allosteric site on PFK1 --> increases affinity for substrate (F6P) and reduces affinity for ATP --> allosteric activator F26BP binds to allosteric site on FBPase1 --> reduces affinity for substrate --> allosteric inhibitor --> slows gluconeogenesis

How does the cell ensure that futile cycles of FA oxidation and synthesis are avoided?

Fatty acid Oxidation (degradation) and reduction (synthesis) are compartmentalized. Synthesis occurs in the cytosol while degradation occurs in the mitochondria.

What reaction controls the entry of glucose carbons into the PPP and how is it regulated?

G6P DH catalyzes NADP+ oxidation of G6P this reaction is regulated allosterically this reaction is inhibited by NADPH and activated by NADP+. The oxidative phase is only active when NADPH is low. It is also regulated covalently when fat synthesis increases or under oxidative stress.

Create a graph of V0 vs [glucose] for both hexokinase and glucokinase include G6P What kind of inhibition might you expect from G6P?

G6P has allosteric inhibition --> cooperative, multi subunit, G6P is the product of both hexokinase and glucokinase if G6P were competitive, it would inhibit both hexokinase and glucokinase. Since only hexokinase is inhibited, it has to be allosterically inhibited. There has to be an allosteric binding site that hexokinase has that glucokinase doesn't have.

What effect would glucagon have on the liver? Would epinephrine have the same effect in the liver?

Glucagon--> converts stored glycogen to glucose that can be released into the bloodstream when blood glucose levels are low, activates Gs --> PKA epinephrine--> breakdown of glycogen to glucose, Gs--> adenylyl cyclase--> cAMP--> PKA

The liver glycogen phosphorylase isoform is allosterically inhibited by glucose when the blood glucose concentration is normal or higher. Why does this make sense?

Glycogen phosphorylase in the liver releases glucose to the bloodstream. If glucose in blood is normal or high, then it doesn't need anymore glucose. Liver maintains steady blood sugar and should only work when blood glucose is low.

The liver isoform of glycogen synthase is also regulated allosterically- in an analogous conformation change to the one glucose induces in the liver glycogen phosphorylase, G6P allosterically activates glycogen synthase. Why does this make sense?

Glycogen synthase in the liver: stores glucose when glucose levels are high in the blood. Only on when blood sugar levels are high. import more and keep phosphorylating. High G6P --> storage options

Isocitrate DH is allosterically inhibited by ATP. This inhibition is relieved by ADP. Why does this make sense?

Goal of TCA-->make ATP if there is a lot of ATP, then more doesn't need to be made because that would be wasteful. If ADP is high, energy status is low and the cycle needs to run.

Consider a pathway that starts with substrate 1 and ends with substrate 4. Substrate 2 is converted to 3 in a far from equilibrium reaction catalyzed by the enzyme E1. Conversion from substrate 3 back to 2 requires a different reaction catalyzed by E2, occurring in the same cellular compartment. Under normal conditions, the activity of E1 is 50pmol of substrate 2 produced per 10^6 cells per second and that of E2 is 40pmol of substrate produced per 10^6 cells per second. What are the direction and overall rate of metabolic flux between substrates 2 and 3?

Goes in the forward direction rate: 10 pmol per 10^6 cells per second of substrate 3 is produced

What are the differences between hexokinase and glucokinase?

Hexokinase--> low Km for sugars ~.1 mM, broad specificity for sugars, inhibited by G6P Glucokinase --> very high K0.5 for sugars (10mM), broad specificity for sugars, sigmoidal dependence on glucose concentration, no inhibition by glucose 6 phosphate

Why does inhibition of hexokinase by G6P make sense? Why does it make sense that glucokinase is not inhibited by G6P?

If G6P gets high, then the flux of glycolysis is slow. The brain is a demand tissue so if G6P gets high then it needs to be inhibited. Only needs the right amount of G6P and doesn't need more. Looks at the needs of the cell now. Works well when glucose levels are low and stops when it doesn't need anymore. The liver is a supply tissue so if G6P gets high then it can go to PPP or glycogen pathways.

Rotenone inhibits the FMN in complex I from accepting electrons. Antimycin A inhibits oxidation of ubiquinol back to ubiquinone by blocking Q sites in cytochrome b. Why are these poisons, and which is worse?

If complex I cant accept electrons, then NADH can't be accepted. NAD+ won't be regenerated. ETC would be slowed because still accept electrons from FADH2 from complex II. --> poison because don't get NAD+ back, blocks TCA and beta oxidation if ubiquinol is not oxidized back to ubiquinone, then ETC is completely stopped. Interferes with FADH2 and NADH --> worse because it blocks all electrons from entering why do brain/skeletal muscle survive longer on rotenone? special shuttle, high efficiency, get electrons from cytosol into matrix

What features of an isoenzyme permit one isoform to be inhibited and another not to be inhibited by the same molecule?

If the regulatory properties such as an allosteric regulatory site is present on one enzyme and not the other, then this can cause inhibition of one isoenzyme but not other other. Enzymes can catalyze different reactions, but are differently controlled by different kinetic parameters and different regulatory properties.

Isocitrate DH and alpha ketoglutarate DH are activated allosterically by calcium. why does this make sense in the muscle?

If there is a lot of calcium, the muscle is contracting. The muscle is using up a lot of energy. Need ATP so need TCA cycle to be running - speed up flux of TCA because the muscle has just contracted and demanding a lot of ATP because it just used a lot of ATP

Glucose is normally completely oxidized to CO2 in the mitochondria. Under what circumstances would glucose be completely oxidized to CO2 in the cytoplasm and how would this occur?

If there is oxidative species present that can create oxidative stress then there is a risk of damage two products of PPP: R5P- nucleotides and NADPH

The muscle glycogen phosphorylase isoform is allosterically activated by AMP. The activation by AMP is blocked by ATP. why does this make sense?

In the muscle, the glucose released by glycogen phosphorylase goes through glycolysis producing ATP (goal of glycolysis is to make ATP) So, if ATP is high enough, activation of AMP will be blocked because more ATP is not needed. AMP- low energy status --> breakdown glycogen --> glucose --> glycolysis ATP- high energy status--> dont need glycolysis runnining

If someone has massive quantities of chylomicrons what is the diagnosis of the patient? What treatment would you prescribe to alleviate the symptoms of this inherited disease?

Lipases are not effectively breaking down triacyclglycerols into fatty acids and glycerol another defect that could result in similar symptoms? apo C II is not functional and this activates lipases normally treatment: don't eat so much fat, reduce dietary fat

List the following in order of increasing electron affinity: cytochrome a +a3, cytochrome b, cytochrome c, cytochrome c1, NADH, oxygen, ubiquinone

NADH --> Ubiquinone --> b--> c1--> c --> a+a3 --> O2

What's the role of NADPH in a cell? When is it produced in high amounts?

NADPH provides reducing power that drives many anabolic reactions that are responsible for biosynthesis of many important parts of the cell such as NT synthesis, lipid synthesis, and nucleotide synthesis. NADPH is the key fuel source for synthesis. When glucose is abundant, NADPH is produced in high amounts through PPP

Oligomycin blocks the proton channel of the F0 domain of ATP synthase. Cyanide inhibits cytochrome a+a3 what would both do to oxygen consumption and ATP production in a preparation of mitochondria? what could you use to distinguish between them?

Oligomycin - prevent ATP production - proton gradient gets high in intermembrane space (cant return to matrix) and oxygen consumption stops Cyanide- oxygen consumption stops- gradient runs to equilibrium and ATP production stops how to distinguish? use an uncoupler, measure the different proton gradients by separating O2 consumption, Oligomycin O2 consumption can still occur in presence of uncoupler because it doesn't require ATP synthesis, cyanide is directly stopped with uncoupler present

Suppose you had the ability to mutate a gene in a specific person. What might you do to to the PTP1B gene in a type II diabetic? why? would this same change impact obesity?

Overactive PTP1B dephosphorylates insulin receptor and IRS stopping the signal reduce expression of PTP1B or less active slow PTP1B --> keep pathway activated a little longer keeps things phosphorylated longer --> helps with diabetes/obesity

Why did DNP kill patients who took the drug to help them lose weight?

Overheat - too much heat released

PDH is stimulated by high levels of pyruvate, NAD+, and CoA but inhibited by NADH and acetyl CoA. Why does this make sense? What would high levels of NADH or acetyl CoA do to the enzymes in the complex?

PDH is activated by substrates --> these accumulate when too little acetate flows into CAC - substrate availability Product inhibition - PDH is inhibited by its products

DCA inhibits the kinase that regulates activity of the PDH complex. Tumor cells show highly increased levels of glycolysis and use lactate fermentation to regenerate NAD+ needed for continued glycolysis, rather than the CAC. what impact would DCA have on pyruvate?

PDH kinase normally inhibits the complex inhibiting the inhibitor --> encouraging pyruvate to be sent through TCA increasing aerobic metabolism --> make less lactate

The use of a citrate pyruvate or citrate malate shuttle allows the continued generation of citrate even if the TCA cycle is inhibited. how?

Passive transporters- metabolic control - concentrations need shuttles to keep citrate synthase running excess citrate to leave then you don't get product inhibition substrate availability - oxaloacetate - need to make more oxaloacetate in matrix bc the TCA is not finishing itself ATP is high - TCA is inhibited

You create an enzyme that can freely use either NAD+ or NADP + in near equilibrium redox reactions. Are you getting fired?

Pools would go to equilibrium if they were used interchangeably NADH and NADPH as separate keep anabolic and catabolic processes happening because there are two separate pools of reducing power

In what ways does regulation of alpha ketoglutarate DH resemble that of PDH?

Same cofactors and multienzyme complex (same E2) --> high energy thioester bonds --> NADH regulated by substrate availability (NAD+ and CoA) regulated by product availability (NADH, succinyl CoA)

F1,6BP is an allosteric activator of pyruvate kinase. Why does this make sense?

The conversion of F6P to F16BP via PFK is an irreversible step that commits the glucose to the glycolytic pathway. Pyruvate kinase generates ATP and pyruvate. If ATP levels are low then F16BP will create a positive feedback loop activating pyruvate kinase. This increases the production of ATP. If energy status is low, AMP binds the allosteric regulatory site on PFK activating it to produce F16BP. F16BP will then activate pyruvate kinase. F16BP speeds up pyruvate kinase so build up doesn't occur. This is feedforward activation that avoids a bottle neck of metabolites.

What is the function of the liver in humans?

The function of the liver in humans is to maintain the blood glucose concentration circulating in the blood stream at reasonably constant levels, in part by converting it to storage forms when blood glucose is high and releasing glucose when blood glucose is low.

What is the role of the liver in glucose metabolism?

The liver controls glucose homeostasis by storing it and being a supply tissue. Controls blood glucose levels.

Concentrations vary by cell type, so the "value" of our currency also varies by cell type. In which cell type is ATP most valuable?

The most valuable cell type would be one with the most negative delta G which demonstrates a favorable reaction ... therefore, we want a small ratio (more reactants than products) the brain is most valuable followed by muscle and then the liver

What is the role of muscle in glucose metabolism?

The muscles regulate glucose homeostasis by being a demand tissue. They maintain energy for contraction.

Consider the following pathways: A: 1--> 2--> 3--> 4--> 5 B: 5--> 4--> 3--> 2--> 1 The metabolic flux of pathway A is now complicated by the fact that molecule 5 could be used as starting materials in pathway B. Describe the possible changes in the flux of pathway A in the presence of pathway B.

Use up 5 to drive B pathway Flux of pathway A needs to increase/be faster because the pathway needs to replace what is being used (5 in this case) to balance out steady state the two steps are running at the same time --> easily adjust the flux if they are both running at the same rate

The two isoforms of glycogen phosphorylase in muscle and liver differ not just in regulation, but differ kinetically as well. Which as a higher Vmax?

Vmax would be higher in muscle because muscle needs energy now, bear Vmax would be lower in liver because glucagon lowers glucose in blood, steady release of glucose into the blood, supply body over slow steady time

PDH is also regulated by phosphorylation/dephosphorylation by kinase/phosphatase components. The activity of the kinase is regulated by ATP and by AMP/ADP. Describe how activity of the PDH complex is regulated by covalent modification and the sensible role allosteric regulation by ATP vs ADP.

When ATP is high, PDH is inactivated by phosphorylation of E1 --> ample fuel is available so don't need anymore, utilizes a kinase, energy status is good so it deactivates it When ADP/AMP are high, PDH is activated by dephosphorylation, utilizes a phosphatase

PFK1 is regulated by ATP, yet ATP is also a substrate. Why does this make sense? how might this work?

When ATP is high, there is a decrease in the affinity of F6P We want PFK1 to stop if ATP is high because the goal of glycolysis is to make ATP. It doesn't make sense to keep running glycolysis if ATP is already high AMP activates PFK1 because energy status is low This could work because there are two binding sites: allosteric/active. Allosteric binds when ATP is high, active site binds when ATP is low. K0.5 should be low at active site, Kd should be high at allosteric site.

a. Predict and draw the oxygen binding curve for pyruvate kinase deficient RBC b. predict and draw the oxygen binding curve for hexokinase deficient RBC

a. favors t state, slows glycolytic flux and favors gluconeogenesis, build up of 1,3 BPG which allows for build up of 2,3 BPG, 2,3 BPG holds T state and shifts curve to the right , slow down last step, everything before it builds up b. If hexokinase is deficient, then 1,3 BPG won't build up and wont have excess be made into 2,3 BPG --> less binding --> curve shifts to the left favoring R state

Where do fats enter the TCA cycle?

acetyl CoA

How does acetyl CoA affect flux through glycolysis and gluconeogenesis?

acetyl CoA activates pyruvate carboxylase encouraging gluconeogenesis and deactivating glycolysis CAC isnt running, out of oxaloacetate increase acetyl CoA --> pre TCA or FA breakdown acetyl CoA binds Pyruvate Kinase to make oxaloacetate using oxaloacetate for gluconeogenesis fuel source --> FA , beta oxidation --> also making ketone bodies to get CoA back

Why might DCA not work?

aerobic structures can also become mutated mutate the binding site decrease PDH and increase affinity of LDH

What is the difference between allosteric and covalent modification in the following reactions? E + ATP --> E*ATP (allosteric) E+ Pi --> E-P (covalent)

allosteric- reversible, nothing off of ATP, noncovalent interaction covalent- covalent bond to a P taken from ATP

What intermediate from the TCA cycle is removed to make glutamate?

alpha ketoglutarate

why would just eating bacon have been a more well rounded choice of a diet?

bacon has protein protein feeds into CAC make proteins in general --> move around / do stuff

What enzymes produce F26BP? What can stimulate an increase in F26BP concentration? What can cause a decrease?

bifunctional enzyme - PFK2 insulin stimulates an increase in F26BP - high blood glucose levels glucagon stimulates a decrease in F26BP- low blood glucose levels

Why does it make sense that glucokinase in liver behaves differently than the hexokinase in other cells?

brain/muscle--> utilize hexokinase--> low intracellular glucose compared to extracellular, needs to be ridiculously high, bring in molecule of glucose and need to immediately phosphorylate it with hexokinase to keep it inside the cell --> keeps glucose in brain/skeletal muscles liver --> utilize glucokinase --> is off at normal glucose levels, on at high glucose levels, lowers glucose if it gets too high, **** glucokinase makes sure that liver cells transform glucose into glycogen and FA if glucose levels are too high, if glucose levels are too low then the low affinity of glucokinase for glucose ensures that the hexokinases of the brain and muscles get glucose first *****

Why would a mutation that reduces activity of the liver enzyme fructose 1,6-bisphosphatase result in abnormally high levels of lactate in the blood plasma?

can't convert F1,6BP to F6P --> everything before this would build up --> pyruvate would build up --> pyruvate goes to fermentation producing lactate acid

Many cancer cells express an alternative isoform of pyruvate kinase (PKM) that has low catalytic activity. Using what you know about cancer cell metabolism, why does this sound wrong? Why is it beneficial to the cancer cells ?

cancer cells increase levels of glycolysis and lactate fermentation to regenerate NAD+ PK is last step of glycolysis to generate Pyruvate from PEP PKM is not producing pyruvate fast enough --> doesn't make sense because they want to use lactate fermentation to regenerate NAD+ and need pyruvate to do that This is actually beneficial because it leads to the build up of G6P which runs to the PPP and get nucleotides for rapid growth gets NADPH for fats and membranes allows the cell to divide and produce more

How would lack of carbohydrates affect your ability to utilize fats?

cant run TCA cycle acetyl CoA --> CAC--> need oxaloacetate run out of oxaloacetate without carbohydrates present

what is the difference between a catalytic coenzyme and a stoichiometric coenzyme?

catalytic- regenerated in complex by itself --> in more than one step --> part of reaction stoichiometric- used and regenerated somewhere else, something else regenerates it, only in one step

How can you change the PFK 1 curve?

change concentration of substrate high substrate concentration - R state low substrate concentration - T state

What intermediate from the TCA cycle is removed to make fats?

citrate (product of citrate synthase)

Citrate inhibits PFK1. This seems sick and wrong, since don't we need to keep glycolysis running to feed fatty acid synthesis? Why does it make sense?

citrate build up = high glycolytic flux PFK1 enzyme for committed step of glycolysis --> reducing glycolysis flow PFK1 inhibition - G6P builds up --> goes through PPP --> get NADPH gives energy needed to get FA synthesis occurring again NADPH through PPP--> get 1/2 of NADPH needed for FA synthesis

Use of the citrate pyruvate shuttle but not the citrate malate shuttle also helps keep FA synthesis running. how?

citrate pyruvate shuttle makes NADPH while citrate malate shuttle makes NADH NADPH is used for FA synthesis NADPH in cytosol through citrate pyruvate shuttle --> get 1 NADPH need 2 NADPH to run FA synthesis

Which enzymes in the TCA cycle are far from equilibrium?

citrate synthase, alpha ketoglutarate DH, isocitrate DH

Succinyl CoA is a competitive inhibitor of citrate synthase. How do you suppose that works, and why does it make sense?

competitive feedback inhibition if succinyl CoA is building up, flux in the back half of the cycle is low. To prevent build up, the activity of citrate synthase needs to be lowered.

what type of cooperativity does PFK1 follow?

concerted

How can allosteric regulation be reversed?

conformational change or energy availability

What effect would you expect epinephrine or glucagon to have on ACC activity? What covalent modification would you expect to occur?

covalent modification - phosphorylation by PKA - low citrate glucagon/epi --> off --> monomer insulin --> on by phosphatase --> filament (dephosphorylation, high citrate)

How to stop allosteric regulation?

decrease concentration below Kd or outcompete metabolic control

Why does it make sense to use a single nucleotide, ATP, as the primary energy currency?

don't want primary energy currency to be too difficult to make --> need to have higher energy options that the cell could use to make more ATP on the fly currency to have enough value to do work --> has high enough phosphoryl transfer potential to activate other metabolites intermediate in energy kinetically stable --> needs enzyme to overcome activation energy barrier

Why do cells pick one molecule for common currency?

easier --> only have to pay attention to one thing and monitor one thing one metric to make sure everything is okay metabolically one energy currency ties all regulation to ATP level

Jolly ranchers on island - eat all at once or one every day?

eat all at once need to shift brain to favor ketone bodies - brain will keep favoring glucose as fuel source as long as you eat one every day and this won't sustain you

If citrate builds up, what does that imply about energy charge? glycolytic flux? what hormone is likely to be in charge in that situation?

energy charge must be high if citrate builds up because there has to be a build up of isocitrate which would be do to increased ATP--> TCA not running to completion glycolytic flux- citrate inhibits PFK1 which reduces the flow of glycolysis --> if citrate is built up, then there doesn't need to be any more energy and so glycolysis can stop running insulin is likely to be in charge because blood glucose levels must be high because glycolysis is happening

Briefly describe how glucagon a/o epinephrine signaling regulates glycogen phosphorylase

epinephrine in muscle and glucagon in liver bind GPCR --> activates adenyl cyclase--> cAMP--> PKA PKA phosphorylates phosphorylase b kinase that phosphorylates phosphorylase converts b--> a turns glycogen synthase off PKA converts A to B inactivating it

The rate of FA synthesis is dependent on the activity of acetyl CoA carboxylase, the cytosolic enzyme that activates acetyl CoA to malonyl CoA. Palmitoyl CoA (16:0) inhibits ACC why?

feedback inhibitor of enzyme look at page

What is meant by an anaplerotic reaction?

figure 16.2

What is flux?

flow of the pathway, moving metabolites through a pathway

The PEPCK gene is mutated resulting in an enzyme that is catabolically inactive. What would happen in gluconeogenesis?

gluconeogenetic flux is stopped --> decrease regeneration of glucose in blood PEPCK catalyzes oxaloacetate in response to glucagon to PEP PEPCK only present when glucagon is present

What is the brains primary carbon source?

glucose to produce ATP The liver needs to maintain blood glucose levels so the brain can have a steady supply of glucose

Shuttle systems are key to ensuring that reducing power collected in the cytosol can be used to generate ATP in the matrix, while also regenerating energy carriers. The compound n-butylmalonate inhibits the malate/alpha ketoglutarate transporter. What happens to glycolysis in muscle cells growing with glucose as their carbon source? what happens to glucose utilization?

glycolysis occurs in cytosol but inhibition of the transporter prevents processes from occurring in the matrix --> cant regenerate NADH to NAD+ need to keep glycolysis running if you can't regenerate NAD+ fermentation --> make lactate ---> what you can do if O2 is low and can't move electrons drastic increase in glucose utilization --> get 2 ATP but normally make 30 more

Would shortening the glycolytic pathway from glyceraldehyde 3-phosphate right to 3-phosphoglycerate benefit the cell?

glycolysis would lose the generation of an ATP this would matter more in an anaerobic because can't ferment because there is not enough energy, and not making any alcohol Aerobic would be fine as long as oxygen is present because you would lose 2 ATP per glucose but would still make 30

Draw the curves of V0 vs [S] curve for F6P draw presence of high ATP/Low AMP and low ATP/high AMP

high ATP/low AMP causes conformational change to favor T state decreasing affinity for F6P acting as an allosteric deactivator low ATP/high AMP more hyperbolic --> high AMP levels activate PFK increasing affinity for F6P, favors R state PFK is sigmoidal because it is an allosteric enzyme that has cooperativity

What distinguishes metabolic from hormonal control?

hormonal control- needs of organism can overcome metabolic control metabolic control- regulator is something in the pathway, level of metabolites in that pathway, needs to cell right now

How can covalent regulation be reversed?

hydrolysis reactions

Pyruvate kinase is not inhibited allosterically by high levels of NAD+. Why?

if anything high levels of NAD+ should activate PK. Increased NAD+, energy status is low --> glycolysis should speed up

Citrate allosterically activates ACC. How might citrate increase Vmax?

increase Vmax need more enzyme active-filament, inactive- monomer citrate binding --> adding more active enzyme --> form filament

What effect would insulin have on the muscle?

increase blood glucose leads to an increase in insulin. An increase in glycogen synthesis occurs by insulin activating PP1 and inactivating GK3 like in the liver. Have a reserve of GLUT4 that are moved to plasma membrane by insulin. Myocytes decrease blood glucose by increasing rates of glucose uptake, glycogen synthesis, and glycolysis

PMS is an artificial electron acceptor. What would happen to TCA cycle flux in the presence of PMS. Why?

increase flux of TCA FADH2 and NADH don't build up never run out of NAD+ or FAD+

it would seem like you would want to eat a lot of sugar at the start of a marathon to increase fuel stores, but experienced marathoners never do this. Why?

increase glucose, increase insulin, glycolysis liver --> glycolysis + FA synthesis --> storage run --> want in supply mode not store mode want glucagon present not insulin

What effect does insulin have on the liver?

increase in blood glucose leads to an increase in insulin. An increase in insulin leads to an increase in PKB which decrease GSK3 which increases glycogen synthase resulting in increased glycogen synthesis. An increase in insulin could lead to an increase in insulin sensitive protein kinases that increase protein phosphatases which increase glycogen synthase resulting in increased glycogen synthesis. Also decrease phosphorylase kinase which decreases glycogen phosphorylase resulting in a decrease of glycogen breakdown. An increase in insulin also increases glycolysis.

What impact would inhibition of isocitrate DH have on the activity of citrate synthase?

inhibition of isocitrate DH would decrease citrate synthase because there would be product inhibition

reverse reaction

intermediates and enzymes are the same

what are branch points?

intermediates might be removed from the TCA cycle to be used elsewhere

The standard free energy change for hydrolysis of ATP is -30.5 kJ /mol. What conditions might be changed to alter the free energy of hydrolysis?

ions like Mg2+ could make ATP more stable affecting the electrostatic repulsion reducing the favorableness of hydrolysis more substrate to drive it forward --> pressure to make it into products, delta G would be more negative and more favorable more products--> delta G is more positive and would be less favorable

Glycolysis

know steps of glycolysis and enzymes what type of reactions are occurring is the reaction far or near equilibrium points of regulation? which reactions are different in gluconeogenesis? which reactions generate energy? what types? which reactions utilize energy?

How would your breath smell if you were on this poor diet?

like acetone, acetone gets exhaled when converting acetyl CoA to ketone bodies --> ketone bodies lower blood pH

When the liver is oxidizing FA to acetyl CoA at a rate that exceeds flux through the TCA cycle, it instead converts the excess acetyl CoA to ketone bodies, which organs like the brain can use as an alternative to glucose. Energetically speaking, what does the liver get out of this deal?

liver would take in glucose to send towards more glucose liver gets CoA back for oxidation of FA --> keeps beta oxidation running to get electrons, still needs to meet liver needs out of oxaloacetate then no TCA

What is the role of glycogen phosphorylase in glucose metabolism in the liver? muscle?

liver- glycogen phosphorylase removes glucose from glycogen, glucose in bloodstream--> emergency fight or flight muscle- muscle needs, glycogen --> last resort storage in muscles --> fight or flight

The isoform of PFK2 found in muscle is not phosphorylated in response to an increase in cAMP. Why is this a very good thing?

look at page

What is the order of electron flow, ending with O2? where does each inhibitor block?

look at recorder sheet 19

What regulates malate DH activity?

malate and NAD+ (substrate availability) oxaloacetate and NADH (product inhibition) if NADH concentration is high then there is high energy status and a lack of oxygen available if oxaloacetate is removed it moves faster

How does malonyl-CoA affect FA oxidation?

malonyl CoA inhibits carnitine acyl transferase and blocks access to transport --> this inhibits oxidation of FA and is used in reduction

amphibolic pathways

metabolic pathways that have both catabolic and anabolic functions

How to stop covalent regulation?

need enzyme- kinase or phosphatase active - need hormonal control to regulate these

If a mouse with a deficiency in producing leptin were parabiotic to a normal mouse, what would happen to the weight of each mouse?

obese mouse will lose weight because set point will match normal mouse normal mouse will stay the same because of their set point decrease leptin initially because increasing blood --> normal mouse will want to eat --> make more leptin --> go back to set point

What intermediated from the TCA cycle is removed to make glucose?

oxaloacetate

Steps 4 and 5 are not necessary for the conversion of pyruvate to acetyl CoA. What is the point of these steps? why are they necessary?

oxidizing reduced lipollysine back to oxidized form of lipoyllysine regenerate lipoyllysine --> cant accept acetyl CoA --> cant get rid of hydroxyethyl group --> cant take in more pyruvate --> gets stuck if oxidation is not regenerated also get NADH - electrons drive oxidative phosphorylation = ATP --> keeps energy status good

Isocitrate DH is also inhibited by NADH. What type of inhibition is occurring? how does this also tie activity to the energy charge of the cell?

product inhibition NADH- high energy electrons If NADH concentration is high, then it doesn't make sense to make more because high energy status lack of oxygen availability

You switch to a diet that is sometimes high in odd chain fatty acids. Is this better than the diet that lack carbohydrates all together?

propionyl CoA is converted to succinyl CoA which can go to CAC get oxaloacetate back yes this would be helpful

What are the advantages of a multi-enzyme complex, like PDH, as opposed to having individual, distinct enzymes that catalyze each reaction of the pre-TCA cycle?

proximity- everything is close, don't have to diffuse far efficient - regenerating ourselves regulation - work as a unit- one unit to worry about substrate channeling - substrate channels through without leaving complex, nothing will get lost, enters and leaves as acetyl CoA

What five enzymes comprise tge PDH complex?

pyruvate DH kinase can inactivate the complex by phosphorylating pyruvate DH pyruvate DH catalyzes steps 1 and 2 (E1) dihydrolipoyl transacetylase catalyzes step 3 (E2) dihydrolipoyl DH catalyzes steps 4 and 5 (E3) protein phosphatase can dephosphorylate the complex and activate it

why is DCA seem beneficial to treating cancer?

pyruvate can be sent through aerobic metabolism instead of fermentation tumor cells--> fast growth, cant control, mutations, invasion a lot of lactate --> lower pH making invasion easier

The pyruvate carboxylase gene is mutated resulting in an enzyme that is catalytically inactive. What would happen in gluconeogenesis?

pyruvate carboxylase converts pyruvate to oxaloacetate gluconeogenic flux is lowered because can't produce oxaloacetate directly from pyruvate however, pyruvate can go through pre TCA and TCA cycle to produce oxaloacetate

Know the difference in steps between glycolysis and gluconeogenesis

regulatory steps of glycolysis are the steps that are different in gluconeogenesis

Reverse pathway

same intermediates but different enzymes - easier to regulate than reverse reactions

Why is it good the citrate moves instead of acetyl CoA?

separate pools of CoA --> key regulatory step, substrate availability Energy status (citrate represents energy status) --> prevent futile cycle from occurring because acetyl CoA is used for FA synthesis and degradation

What are the two types of cooperativity?

sequential--> in between concerted--> all at once

What chemical factors make ATP high energy? (large negative free energy change?

stabilization of the products through resonance--> more resonance in products (stable) and less resonance in reactants (unstable) greater solvation of the products than the reactants --> really hydrophilic --> products can bind more water **** biggest contributor **** Relief of electrostatic repulsion between phosphates --> 4 negative charges clustered together in the 3 phosphates, increase potential energy of bonds, more energy if you are unstable

How do stand and actual free energy changes differ?

standard free energy change- directly related to the equilibrium constant. The standard free energy change tells us in which direction and how far a given reaction must go to reach equilibrium when the initial concentration of each component is 1 M, the pH is 7, the temperature is 25 degrees C, and the pressure is 1 atm. Standard free energy change is a constant. actual free energy change- a function of reactant and product concentrations and of the temperature prevailing during the reaction that might not match standard conditions

rate determining step

the slowest step in a reaction mechanism the step in an enzymatic reaction with the greatest activation energy or with the transition state of highest free energy

reciprocal regulation

upregulate one and downregulate the other same molecule regulates in both directions

Pyruvate kinase in liver is phosphorylated as a result of a cAMP signaling cascade. The muscle isoform is not phosphorylated. What effect would you predict phosphorylation should have on PK activity, and why does it make sense that the two tissues have different isoforms?

want to be able to turn off glycolysis in the liver but not in the muscles (need fight or flight response) phosphorylation deactivates molecule --> wont catalyze reaction --> liver does not need glucose as much as brain/muscle, if there are low amounts of glucose in the blood, PK will be deactivated to ensure glucose molecules are not being taken up by liver cells