Glycolysis and Gluconeogenesis

How does glucose enter the bloodstream?

via the breakdown of polysaccharides or synthesis by non-carbohydrate precursors

How is glucose stored locally in the muscles?

glycogen

How many reactions are part of the glycolysis pathway?

10

What is the net generation of ATP from glycolysis of 1 glucose molecule?

2 ATP

Where does glycolysis occur?

cytosol

Which steps of glycolysis are part of the investment stage? What has to be invested?

1-5; 2 ATP has to be invested to drive glycolysis in one direction

What is reaction 1 of glycolysis?

glucose + Mg2+ + ATP <--> G6P + ADP via hexokinase

Where is hexokinase IV (glucokinase) found?

liver

Does hexokinase IV have higher or lower affinity for substrates than hexokinase I, II, and III?

lower (higher KM than I-III)

Why is magnesium needed for hexokinase activity?

it shields the 2 negative charges from the first 2 phosphate groups of ATP, making the gamma phosphate susceptible to nucleophilic attack by C6-OH of glucose

Why is the first reaction of glycolysis a good example of the catalysis by proximity effect?

when glucose binds to hexokinase, there is a conformational change that brings glucose close enough to ATP for the nucleophilic attack

What is the second reaction of glycolysis?

G6P <--> F6P via phosphoglucose isomerase (PGI)

What is the mechanism of the second reaction of glycolysis?

general acid-base catalysis; isomerization of an aldose to a ketose

What is the third reaction of glycolysis?

F6P + ATP + Mg2+ --> F1,6BP + ADP via phosphofructokinase-1 (PFK-1)

Why is the third step of glycolysis the rate-limiting step?

PFK-1 is the rate-limiting enzyme of glycolysis

What allosterically activates PFK-1?

AMP

What allosterically inhibits PFK-1?

ATP and citrate

What is the fourth reaction of glycolysis?

F1,6BP <--> 2 GAP/DHAP via aldolase

What type of reaction is the fourth reaction of glycolysis?

retro aldol condensation (aldol cleavage)

Why does G6P have to be converted into F6P and then F1,6BP before cleavage occurs?

aldol cleavage of G6P/F6P would yield products of unequal length (aldol cleavage requires a carbonyl on Cn and a hydroxyl on Cn+2)

What is the fifth reaction of glycolysis?

DHAP <--> GAP via triose phosphate isomerase (TIM); aldose-ketose isomers

What is the intermediate between the conversion of GAP and DHAP?

enediol

What is the mechanism of the fifth reaction of glycolysis?

acid-base catalysis

How has TIM achieved catalytic perfection?

the rate of reaction is diffusion controlled and product formation occurs as rapidly as substrate comes into contact with TIM the enzyme

What happens to [GAP] and [DHAP] if the cell is in a steady state?

[DHAP] increases and becomes greater than [GAP]

What happens in the energy-recovery stage of glycolysis? Which reactions are part of this pathway?

low-energy reagents get transformed into high-energy products able to form ATP via substrate-level phosphorylation; 6-10

What is the sixth reaction of glycolysis?

GAP + NAD+ + Pi <--> 1,3-BPG + NADH via glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

What type of reaction is the sixth reaction of glycolysis?

aldehyde oxidation

How does the sixth aldehyde oxidation reaction take place if it has a positive ∆G?

it is coupled with the seventh reaction

What is the relationship between the energy of GAP and 1,3-BPG in the sixth reaction of glycolysis?

GAP has lower energy than the product 1,3-BPG

What is the seventh reaction of glycolysis?

1,3-BPG + ADP <--> 3PG + ATP via phosphoglycerate kinase (PGK)

What happens to the 1,3-BPG product in erythrocytes and why?

it becomes 2,3-BPG because when 2,3-BPG binds to deoxyhemoglobin, it reduces the affinity of hemoglobin to O2, allowing oxygen to be delivered to tissues

What is the eighth reaction of glycolysis?

3PG <--> 2PG via phosphoglycerate mutase

What is the ninth reaction of glycolysis?

2PG + Mg2+ <--> phosphoenolpyruvic acid (PEP) + H2O via enolase

What type of reaction is the ninth reaction of glycolysis?

dehydration

What is the most energetic compound of glycolysis?

phosphoenolpyruvic acid (PEP)

What is the tenth reaction of glycolysis?

PEP + ADP --> pyruvate + ATP via pyruvate kinase

How is ATP formed in the tenth reaction of glycolysis?

pyruvate kinase couples the free energy of PEP breakdown to form ATP and pyruvate

What is fermentation?

generation of ATP without consuming O2 or NAD+

Is fermentation favorable?

yes; it is very energetically favorable with a thermodynamic efficiency of >50%

Where and when does homolactic fermentation occur?

in the muscles when there is high muscle activity

What is the reaction that takes place in homolactic fermentation?

pyruvate + NADH <--> lactate + NAD+

Once lactate is produced, what can happen?

it can stay in the cell to get converted back into pyruvate OR exported to the liver to help synthesize glucose

What does acidification of a muscle prevent?

its continuous, strenuous work

Where does lactate go after building up in the muscle?

to the Cori Cycle in the liver, where it gets converted into glucose or eventually glycogen

When and where does alcoholic fermentation occur?

in yeast under anaerobic conditions

What is the reaction that takes place in alcoholic fermentation?

pyruvate --> acetaldehyde + CO2 via pyruvate decarboxylase and then acetaldehyde + NADH --> ethanol + NAD+ via alcohol dehydrogenase

What is the Pasteur effect?

yeast consume much more sugar in anaerobic conditions than in aerobic conditions

Why does the Pasteur effect occur?

more glycolysis is needed for anaerobic respiration than aerobic respiration

In which organs does gluconeogenesis occur?

liver and kidneys

What are the 4 enzymes that differ between glycolysis and gluconeogenesis?

pyruvate carboxylase, PEPCK, fructose-1,6-bisphosphatase, and glucose-6-phosphatase

What is the net energetic cost of gluconeogenesis?

6 ATP equivalents (2 each for PEPCK, pyruvate carboxylase, phosphoglycerate kinase)

What type of prosthetic group does pyruvate carboxylase have?

a biotin prosthetic group that carries CO2

How do we determine how glycolysis is regulated?

look at rate-determining steps (1, 3, and 10), allosteric modifiers, and measuring proposed regulators

What enzymes are regulated in glycolysis?

hexokinase IV (glucokinase), PFK-1, and pyruvate kinase

Why is hexokinase IV not the rate-limiting enzyme of glycolysis?

it is bypassed in the muscles when glycogen is converted directly into G6P

Why is pyruvate kinase not the rate-limiting enzyme of glycolysis?

it is the enzyme used in the last step of glycolysis

What is the major flux-controlling enzyme of glycolysis in muscle?

PFK-1

What type of enzymes in a pathway limit flux of metabolites more than others?

regulated enzymes (far from equilibrium)

Increased hexokinase activity enables activation of ______________.

glucose

Increased PFK-1 activity enables catabolism of _________________.

glucose

Which enzyme regulates flux in glycolysis more?

hexokinase IV

What activates PFK-1?

AMP, ADP, F2,6P

What is the most powerful allosteric effector of PFK-1?

F2,6P

What inhibits PFK-1?

ATP, citrate

What 2 sites does each subunit of PFK-1 have?

substrate and regulative

What does ATP bind to in PFK-1?

both the substrate and regulative sites, but it freezes the regulative site in the T position, making it unable to bind F6P anymore

What is the mechanism of ADP binding to PFK-1?

it prevents the unwinding of Arg 162, which stabilizes the R state

What is substrate cycling?

the control process in which opposing reactions are catalyzed by different enzymes

What is a futile cycle? What is a good example of a futile cycle?

two opposing reactions occur simultaneously, resulting in a net loss of energy; third reaction of glycolysis combined with the ninth reaction of gluconeogenesis

What does high F2,6BP activate and inhibit? What is the result?

it activates PFK-1 and inhibits FBPase; glycolysis proceeds

What does low F2,6BP activate and inhibit? What is the result?

it activates FBPase and inhibits PFK-1; gluconeogenesis proceeds

What produces F2,6BP? What eliminates it?

PFK-2 produces it and liver FBPase-2 eliminates it (enzymes are part of same homodimeric protein)

What happens to F2,6BP if there is high insulin in the blood?

high insulin activates PFK-2, which increases F2,6BP, which activates PFK-1, promoting glycolysis

What do high levels of acetyl CoA activate? Why?

pyruvate carboxylase; to increase oxaloacetate for the citric acid cycle

Does PEPCK have any allosteric regulators?

no