Glossary of 4. Glycolysis and Gluconeogenesis

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What is glycolysis? What are its 2 possible products? By what pathways?
Metabolism of sugar to:
-2 Pyruvate (aerobic)
-2 Lactate (anaerobic)
What is the net energy yield per glucose molecule in glycolysis?
What is the great advantage of glycolysis?
Can run at high speed, and
Can run in absence of oxygen at expense of flooding tissues w/ lactate.
What in general terms is gluconeogenesis?
Replenishing the glucose consumed by glycolysis.
What is the purpose of gluconeogenesis in the liver?
To maintain blood glucose levels
What is the purpose of gluconeogenesis in muscle?
To replenish muscle glycogen consumed in earlier bursts of muscle activity.
How far back in reverse of glycolysis does muscle gluconeogenesis proceed? Why?
Only back to G6P - muscle does not have G6Phosphatase.
What exactly is produced by glycolysis?
-2 Pyruvate
-2 ATP
Where does pyruvate go after glycolysis?
If O2 is available, to TCA.
In what cell compartment does glycolysis take place?
Where does gluconeogenesis occur?
Partly in cytosol
Partly in mitochondria
What do isomerases do?
Change aldehydes to carbonyls and vice versa
What do mutases do?
Move phosphates
Is glucose the only sugar substrate for glycolysis?
-Fructose enters directly
-Others (galactose) enter after conversion.
Why is glucose the ideal substrate for rapid energy production vs FA's which have more energy content?
-It is more soluble
-Can be metabolized when O2 is limiting
What tissue does gluconeogenesis mainly occur in?
Why doesn't as much gluconeogenesis happen in muscle?
They don't have the enzymes necessary to generate free glucose
What is "gluconeogenesis" (not really) used for in muscle?
To replenish GLYCOGEN.
What are the 2 main stages of glycolysis?
1. Making two 3-carbon molecules from one 6-carbon glucose.
2. Using the 2 products of stage 1 to generate 2 ATP total
Why are only 2 ATP generated in glycolysis?
-2 invested in stage one
-2 produced per 3-C molecule (4)

What begins glycolysis
Historic: includes glucose trapping via hexo/glucokinase

Formal: PFK-1 catalyzed reaction (rate determining step of pthwy)
What are the 2 irreversible steps in glycolysis?
1. PFK-1 (commitment step_
2. Pyruvate kinase (last step)
How many enzymes are involved in glycolysis?
9 (not including the glucose trapping enzyme hexokinase)
What are the 9 enzymes?

Pumpkin Pie Apple Tart
Grateful Phor Plenty of Enzyme Pathways
PPATGPPEP stands for:
1. Phosphoglucose isomerase
2. Phosphofructokinase
3. Aldolase
4. Triose phosphate isomerase
5. G3P dehydrogenase
6. Phosphoglycerate kinase
7. Phosphoglycerate mutase
8. Enolase
9. Pyruvate kinase
What are the enzymes for the irreversible steps of glycolysis?
1. 'Pie' - phosphofructokinase

9. 'Pathways' - Pyruvate kinase
What is the first step of glycolysis?
An isomerization to convert G6P to F6P
How is the isomerization done?
Via simple acid-base catalysis.
What enzyme is very important in glycolysis? Why?
B/c it catalyzes the rate-determing step of commitment.
What happens in the reaction catalyzed by PFK1?

What is the product?
-Another phosphate is donated from ATP to Fructose6Phosphate

-Generates F-1,6-Bisphosphate
What is the major difference between glucose and fructose (achieved by an ISOMERASE)?
Isomerases convert aldehydes to ketones (glu->fru).
What is the quatern. structure of PFK1?
What is involved in the PFK reaction?
ATP hydrolysis
At what carbon is Fructose 6-P phosphorylated by PFK1?
What is the anomeric carbon?
Carbon 1

Carbon 2 is anomeric
What type of regulation is PFK1 subject to?
How is PFK1 exist?
In one of 2 conformations:
R-relaxed, active

T-taut, inactive
What conformation is preferentially bound by
Activators - bind relaxed

Inhibitors - bind taut
What is the main allosteric inhibitor of PFK1?
ATP - acts as a general signal of the cell's energy content; if high, indicates no need for E.
How does ATP alter PFK1?
By altering its Km for F6P:
-High ATP = High Km of F6P
-Low ATP = Low Km...
How does ATP alter PFK's activity as Fructose-6-P conc. increases on a graph?
Sigmoidally - only as F6P substrate increases does the inhibitory effect of ATP get overcome.
What is the effect on PFK1 activity when both AMP+ATP are present?
AMP increases it - not as much as if ATP were not there, but it makes PFK1 active even at low F6P concentrations. (graph)
What are the 3 allosteric inhibitors of PFK1?
1. ATP
2. Citrate
3. Low pH (acid)
Why is citrate a PFK inhibitor?
B/c it is TCA cycle intermediate.
Why is Acid an inhibitor of PFK?
It occurs when Active glycolysis generates Lactic Acid (pyruvate not converted to CO2).
What are the 3 energy metabolite allosteric activators of PFK1? How do they work?
AMP, ADP, and Pi;
Bind allosteric ATP site, so they compete with ATP.
What is a non-energy metabolite allosteric activator of PFK1?
Fructose 2,6-Bisphosphate
How does F26BP alter the activity of PFK1?
By decreasing its Km; Thus PFK1 operates more efficiently even at low F1,6BP concentrations.
How exactly does F2,6BP decrease the Km of PFK1?
Binding increases PFK1's affinity for substrate (F6P) and decreases ATP's inhibition.
Why is Fructose 2,6BP so important?
B/c it is NOT a metabolic intermediate, but a longer term regulator; it sets the rate of glycolysis. MOST IMPORTANT!!!
What is F26BP itself regulated by?
Insulin and glucagon levels in the liver.
How is the regulatory molecule F26BP made? What enzyme?
By conversion of a little bit of F6P by the enzyme PFK2

What enzyme breaks down F26BP when activation is not desired?
Fructose Bisphosphatase-2 (FBPase-2)

How are PFK2 and FBPase-2 related?
They are both the same enzyme, just bifunctional - reciprocal kinase /phosphatase activities.
To what 2 types of regulation is this bifunctional enzyme subject?
1. Allosteric (via F6P)
2. Covalent
Where does covalent regulation of PFK2/FBPase-2 take place? How?
(for the liver enzyme)
-At a single serine residue in the gene regulatory domain;
-Gets Phosphorylated by a Protein Kinase.
So what are the 3 domains of the PFK2/FBPase-2 gene?
1. Phosphatase
2. Kinase
3. Regulatory
What directs covalent regulation?
Insulin vs. Glucagon levels in the liver.
How does phosphorylation of the Ser on the regulatory domain of the bifunctional enzyme alter glycolysis?
Phosphoryltn = FBPase2
(no glycolysis b/c no PFK1)

DEPhosphorylation = PFK2 (Glycolysis b/c PFK1 = active)
What does high Glucagon stimulate?
Increased cAMP; Active cDPK; Dephosphorylation so PFK2 is inactive and glycolysis WON'T occur - THE LIVER NEEDS TO RELEASE GLUCOSE AND MAKE IT; not break it down.
What concurrently results when Glucagon stimulates FBPase2 formation by phosphorylating the regulatory domain?
Stimulation of gluconeogenesis.
What step takes place after PFK produces Fructose 1,6-BP?
It is cleaved into DHAP and G3P (GAP) by Aldolase
What type of reaction is catalyzed by Aldolase?
The reverse of an aldol condensation - produces an aldehyde (GAP) and ketone (DHAP)
Which product of aldolase continues with glycolysis?
Only GAP
How is DHAP converted to GAP - by what enzyme?
Triose phosphate isomerase (TIM)
What would be the result of no TIM were present?
Only 1/2 of each glu molecule would proceed thru glycolysis - no net production of ATP.
What is the 1st step of the energy-producing part of glycolysis? Why is it special?
Conversion of GAP-> 1,3-BPG
-First oxido-reduction
-Generates hi-energy PO4 bond
What 2 things account for the energy derived in this conversion?
-Hi energy phosphate bond
-NADH produced
How does G3P-dehydrogenase allow the hi-energy PO4 bond to be made?
Has a Cys in active site; oxidizes aldehyde of GAP to C=O; Phosphate attachment yields MIXED ACID ANHYDRIDE
What is the use of a mixed acid anhydride?
It drives the formation of ATP via substrate level phosphorylation in the next step.
So why is the GAP-> 1,3BPG step special?
1. First redox reaction
2. Generates hi-energy bond
What inactivates GAP dehydrogenase? (2 things)
1. Heavy metals - inactivate the thiol in active site.
2. Arsenate
How does Arsenate inhibit GAP dehydrogenase?
By mimicking Pyrophosphate; prevents forming the hi energy bond, and ATP generating step is skipped.
What enzyme catalyzes the step that harvests the energy of the mixed acid anhydride 1,3-BPG?
Phosphoglycerate kinase
What are the products of this harvest?
ATP + 3-Phosphoglycerate
What happens after the first substrate level phosphorylation?
Mutation of 3-phosphoglycerate; PO4 moves to Carbon 2
What enzyme catalyzes this mutation?
Phosphoglycerate mutase
What step occurs after 2-phosphoglycerate is formed?
Dehydration - removal of an OH to form a double bond
What catalyzes this dehydration step 8, and what is the product?
Enolase - produces PEP (phosphoenolpyruvate)
Why is PEP so peppy?
Because it has a PHOSPHATE coming right off a double bond; that's very unstable.
What is the use of the phosph-enol of PEP?
Provides energy for another substrate level phosphorylation.
What is the final product of glycolysis?
What enzyme catalyzes formation of pyruvate?
Pyruvate kinase.
What is an alternative use of 1,3-BPG rather than the first substrate level phosphorylation?
Can be converted by RBCs to 2,3-DPG via BPG mutase.
What regulates the concentration of 2,3-DPG in rbcs?
Rates of Mutase and Phosphatase
What does 2,3-BPG phosphatase form?
3-phosphoglycerate for re-entry into glycolysis.
Is the 2nd substrate-level phosphorylation reversible?
What regulates pyruvate kinase?
F1,6BP - it is an allosteric feedforward activator.
Why is it good that F16BP is an activator of PK?
Because it allows the 2 regulatory steps to be coupled.
What are 2 feedback inhibitors of PK?
Why is ALANINE a feedback inhibitor of PK?
-High levels signal protein degradation; meaning blood glucose and glycogen levels are depleted; now the liver should be doing gluconeogenesis.
How are Alanine and Pyruvate related to each other?
Structurally; can be interconverted via an aminotransferase.
What type of regulation is LIVER pk subject to?
Covalent - cADPK can phosphorylate it.
What do you think happens to glycolysis when cADPK phosphorylates PK?
cADPK would be stimulated by glucagon which sends a signal to stop breaking down sugar in the liver, and instead run gluconeogenesis to replenish the blood glucose.
So phosphorylated PK is ____ and
unphosphorylated PK is ____?

What enzyme dephosphorylates PK to reactivate it?
Phosphoprotein phosphatase
How does covalent regulation of PK relate to that of PFK1?
Phosphorylated bifunctional enzyme has FBPase1 activity; F26BP levels decrease and PFK1 is not active.
During ANAEROBIC glycolysis how is NAD+ regenerated?
By converting Pyruvate to lactate (concomitant oxidation of NADH)
During AEROBIC glycolysis how is NAD+ regenerated?
By oxidative phosphorylation in mitochondria.
2 Shuttles regenerate NAD+ for aerobic glycolysis:
1. Glycerol phosphate shuttle

2. Malate Aspartate shuttle
How does the Glycerol phosphate shuttle work?
G3P dehydrogenase on inner mito membrane oxidizes G3P (diffused thru outer mito memb) by reducing FAD to release FADH2 into mitosol.
What gets produced from this?
DHAP - into the mito intermembrane space; diffuses back to cytosol
What happens to the FADH2 generated in the mitosol?
It goes on to ETC
What is the drawback to relying on the Glycerol phosphate shuttle for NAD+ replenishing?
Oxidative phosphorylation only produces 1.5 ATP/pair of reducign equivalents transferred from NADH to FADH2 utlimately.
How does the Malate-OAA shuttle work?
1. OAA is reduced to Malate by cytosolic malate dehydrogenase - produces NAD+ in cytosol.
2. Malate exhcanges for a-ketoglutarate to get into mito matrix.
3. Malate transfers e- to NAD+ by mitochondrial malate dehydrogenase + OAA
How does OAA get back out of the mito matrix to cytosol?
Converts to 4-Carbon Asp which can cross; Glu brings amino group back into matrix.
What determines how fructose enters glysolysis?
Tissue type:
Other than liver: hexokinase directly phosphorylates fructose
Liver: glucose outcompetes fructose for the active site of glucokinase.
What pathway takes fructose into glycolysis in the liver?
Fructose-1-phosphate pathway; requires ATP, Fructokinase, Fructose 1-phosphate aldolase, Triose Kinase, and G3P.
What is required for the F1P pathway in liver? (substrate adn 3 enzymes)
-Fructose (duh) + ATP
-Fructose 1-phosphate aldolase -Triose Kinase
What is a good/bad thing about fructose entering glycolysis?
It bypasses the regulatory step of PFK1 b/c it enters as G3P.
Why is it bad for Fructose to enter glycolysis if high?
It ties up all the phosphate and depletes ATP.
How does Galactose enter glycolysis?
As glucose-6-phosphate
What is the key enzyme that converts galactose to glucose?
An epimerase - flips one hydroxyl to make glucose.
So how does Glucagon exert its effect?
Via cAMP-depenent protein kinase
What are the sites of action of cAMP-dependent protein kinase in regulating glycolysis? What signal does it send?
-Pyruvate kinase
All are inhibited by glucagon
What are the majority of enzyme defects in glycolysis related to?
Pyruvate kinase
How is enzyme deficiency in glycolysis mainly manifested?
As HEMOLYTIC ANEMIA - RBCs can't mantain adequete ATP to maintain their membranes.
What 4 molecules can glucose be MADE from?
-Glycerol (lipid backbone)
What is the difference between glucogenic and ketogenic amino acids?
Glucogenic - can become glucose

Ketogenic - only lead to AcCoA; can't become glucose. (Leu/Lys)
What are the 4 enzymes unique to gluconeogenesis b/c they are involved in irreversible steps?
1. Pyruvate carboxylase
2. PEP Carboxykinase
4. G6Pase
What is the first step in gluconeogenesis?
Generation of PEP from pyruvate.
Where does pyruvate come from?
The mitochondrion
What enzymes are needed to bypass the irreversible step of Pyruvate->PEP?
-Pyruvate carboxyylase
-PEP Carboxykinase
What is BIOTIN?
a coenzyme derived from the vitamin Biotin (aka Vit H)
Which enzyme has the biotin coenzyme? Where is this enzyme?
Pyruvate Carboxylase - located in mito matrix.
What reaction does Pyruvate Carboxylas catalyse?
Pyruvate + CO2 (AT) -> OAA (ADP+Pi)
What are the 2 steps of the Pyruvate Carboxylase reaction?
1. Activate enzyme via CO2 addition *Requires ATP
2. Lose Enzyme, Add Pyruvate to CO2; makes OAA
How does mitochondrial OAA get into the cytosol?
Mito Malate dehydrogenase converts OAA to Malate by oxidizng NADH -> NAD; malate can diffuse.
What happens to malate in cytosol?
Reconverted to OAA (produces NADH)
What acts on OAA in the cytosol?
PEP Carboxykinase - uses a GTP to make high energy PEP and lose CO2.
The pathway using malate as the shuttle for OAA from mito to cyto is probably wrong; what is right?
OAA + Glu -> Asp + a-KG (mito)
-Asp/a-KG diffuse to cytosol-
Asp + a-KG -> OAA + Glu
-Glu takes NH3 back to mitosol-
Why is the latter shuttling pathway probly correct?
It doesn't generate NAD+ in the mitosol or consume NAD+ in the cytosol.
What happens after PEP is formed in the cytosol?
The next steps of glycolysis are simply reversible, up to PFK1
What allows for glycolysis to simply be reversed? (2 things)
-Law of mass action - the elevated levels of PEP push reactions backward.
-Liver Pyruvate Kinase is inactivated by PKA during gluconeogenesis
What enzyme catalyzes the step in place of PFK1 (which can't go backwards)?
F1,6BPase - a critical site of gluconeogenesis regulation!!!
What allosterically inhibits F1,6-BPASE?
AMP - signals low energy; want glycolysis
What also inhibits F1,6BPase?
-Inhibitor of gluconeogenesis
-Activatory of glycolysis
What is the last irreversible step of glycolysis that must be overcome?
Conversion of G6P to free glucose + Phosphate
Where is the enzyme for reversing hexo/glucokinase?
Inside ER lumen: Glucose-6-phosphatase (G6Pase)
What tissue is rich in G6Pase?
Liver (not muscle). B/c glucose levels fluctuate a lot more in liver than in muscle.
What happens to skeletal muscle in strenuous exercise?
It doesn't have enough O2 to run aerobic glycolysis -> produces lactate.
What happens to the lactate generated from exercising muscle?
Goes to blood, then liver.
What does the liver do with lactate?
Converts it to pyruvate then glucose
How much ATP is required for the liver to make 1 glucose?
What is this cycle (Muscle producing lactate sent to liver producing glucose sent back to muscle) called?
The Cori Cycle

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