Glycolysis Flashcards
what tissues contain GLUT 1? what is its KT and how does this relate to the function?
all RBC
1mM - low KT due to high affinity needed for constant uptake of glucose
where is GLUT 2 expressed? what is its affinity and how does this relate to its function?
in the liver, pancreas and inststine
15-20mM- lowest affinity (highest KT)
-this transporter only comes into play when blood glucose is high
where is GLUT 3 expressed? what is its KT and how does this relate to its function?
in the brain
lowest KT (highest affinity) due to large glucose demands
where is GLUT 4 expressed? what is its affinity and how does this relate to function?
In the muscle, fat and heart
-5mM: only present when glucose levels are high
-regulated by insulin
what are typical blood glucose levels?
5mM
Where are GLUT 5 transporters expressed? what do they have a high afinity for?
in epithelial cells
- high affinity for FRUCTOSE and low affinity for GLUCOSE
explain how GLUT 4 activity is affected by insulin?
1) insulin will bind to receptor causing conformation change
2) The activated insulin receptor, through a series of other messengers, causes the exocytosis of vesicles containing GLUT 4 to the cell memnrane
3) Glucose can then enter the cell from the bloodstream through diffusion
In the muscle, heart and adipose tissue
where is GLUT 4 stored when insulin is not present?
inside vesicels of the cell
-adipose, heart and muscle tissue cells
what would you expect GLUT 4 receptors to be like in endurance athletes?
they will have higher levels of GLUT 4 to better deal with increased sugar levels
what is the reduced pathway of pyruvate?
conversion to lactate
what is the net oxidation of fermentation?
There is no net oxidation of fermentaion
- it is the anaerobic breakdown of nutrient molecules (lactate / ehtanol fermentation)
what is the role of phosphorylation in the energy investment phase of glycolysis?
1) activate the product
2) regulates [glucose] into the cell
-keeps levels low so we can continue to phosphorylate it
what is step 1 of glycolysis, how is it regulated? what is the enzyme that carries out this rxn?
-phosphate transfer
-regulated by product inhibition
-carried out by hexokinas
what kind of bond is formed from the phosphorylation of glucose?
phosphoester
-not high energy
what is the dela G for the formation of a phosphoester bond?
~ +16kj/mol
what is the significance of Mg2+ with ATP?
it reduced the (-) charge on oxygen to allow the bond to be broken
If glucose is not present in a catalytic site, will ATP bind? why or why not?
No, it will not bind because:
1) water could potentially hydrolyze ATP
2) there would be no conformational change and ATP may not fit
-making the use of ATP a waste
what are isozymes?
different proteins that catalyze the same reaction
what is hexokinaseā isozyme? How do they differ?
Hexokinase:
-found in many cells
- less specific (can phosphorylate many 6C molecules)
- product inhibited
- low kM (high affinity)
- hyperbolic
Glucokinase:
-found in liver and pancreatic cells
-specific
-regulated by compartmentation
-high kM (lower affinity)
-sigmoidal
why does glucokinase have a lower affinity for glucose?
it has to be able to sense smaller changes in blood glucose levels and reulate insulin levels proportional to glucose levels
what does glucokinase do?
conversion of glucose to G6P in the liver and pancreas
explain the mechanism for insulin release?
In the pancrease:
1) Glucose enters the cell through GLUT 2, propertionaly to the amount of glucose digested
2) Glucose then goes through glycolysis, being catalyzed by glucokinase
3) ATP produced from glycolysis will inhibt K+ eflux from the cell
4) the decreased K+ outside of the cell will depolarize Ca+ channels
5) depolarization of Ca+ channels willopen them, causing Ca+ to move inside the cell
6) increased Ca+ in the cell will stimulate the release of insulin into the bloodstream
https://youtu.be/5SsS_PCrDTo?si=AeryZrwM8inZo6Pj
how is glucokinase (GK) regulated in the liver?
1) When blood glucose levels are low, GK is bound to glucokinase receptor protein (inactive state) in the nucleus
2) as blood glucose levels rise, GLUT 2 is insterted into the cell membrane and glucose levels rise in the cell
GK can be released from GKRP in two ways:
a) Fructose-1-phosphate is metabolised in the liver from ingested fructose which aids in the displacement of GK from GKRP
b) Direct binding of glucose to GK causing conformational change and releasing it from GKRP
3) the free GK moves to the cytosol where it binds with glucose and is converted to G6P
4) glycolysis is then carried out and glucose / fructose levels decrease and GK is bound back to GKRP
when glucose is converted into G6P in the liver, what is the first thing that will be done with G6P?
G6P will be converted to glycogen to ensure stores are full
Where is the major site for fat production?
the liver
what is phosphoglucose isomerase (PGI)?
The enzyme responsible for opening the Glucose-6-phosphate ring, catalyzing the isomerization and closing the ring
What is the most important regulatory step of glycolysis? what kind of rxn is it? what enzyme catalyzes this?
the conversion of F6P to
F1,6-BP
-PFK-1 tightly regulates it
-phosphate transfer
why is the rxn catalyzed by PFK-1 the commited step of glycolysis?
after the conversion of F6P to F-1,6-BP there are no other main pathways that can be taken
-not always commited to glycolysis even though this is the case
what reaction contains the cleavage to two triose phosphates? what enzyme catalyzes this?
the lysis rxn of F-1,6BP
-aldoase
how is the lysis reaction to DHAP and GAP kept going forward if it is reversible?
low [product] due to the constant usage prevents it from going in reverse
what enzyme catalyzes the isomerization from DAP to GAP?
Triose phosphate isomerase
what makes 1,3-BPG an energy rich compound?
it contains a mixed anhydride bond which is very high energy
-during the hydrolysis of an acyl phosphate, both products are stablilized by reosnance which contributes to its high energy
what is the importance of phosphoglycerate kinase (PGK)?
it is the ezyme that catalyzes a substrate level phosphorylation for 1,3-BPG to 3PG
what kind of enzyme catlyzes the transfer of a functional group in the isomerization of 3PG to 2PG?
a mutase
-PGM
how do isomerizations affect oxidation states ?
usually does not change them
what enzyme catalyzes the generation of PEP? what kind of molecule is PEP?
enolase generates the high energy molecule PEP
in addition to PEP, what else is produced from the generation of this high energy intermediate?
water
what is the first structure generated from the substrate level phosphorylation of PEP? what is the final product and how is it formed?
an unstable enol form of pyruvate is formed which almost instantly tautomerizes to form pyruvate
How does the strcuture of alanine differ from pyruvate?
they differ in the addition of a amino group on alanine
what are the high energy intermediates of glycolysis?
1,3-BPG and PEP
what do you expect the delta G to be for the first and third rxnās of glycolysis? (Hexokinase and PFK-1)
they are largely negative due to them being irreversible
what other pathway can 1,3BPG take from glycolysis?
it can be pulled out of glycolysis in RBC and used to make 2,3-BPG
what is the function of 2,3-BPG?
binds to the central cavity of hemoglobin to reduce affinity for oxygen
what occurs in erythrocytes with a hexokinase deficiency? why?
reduction in hexokinase will prevent conversion of Glucose to G6P and reduce the continuation of glycolysis
- this will prevent the productoin of 1,3BPG as a byproduct
- less 2,3BPG production
- decreased binding to hemoglobin which will increase affinity of hemoglobin for O2
how do you get mannose in the body to be used in glycolysis?
in proteins ingested. Is not present in the diet, but will be in chains of sugars that are attached to proteins
- comes into the cycle at F6P (substrate for hexokinase and glucokinase)
what part in gycolysis does galactose come in? what enzyme is used?
at G6P
- galactokoinase
when does fructose come into glycolysis? what ezyme is used? where?
fructose in the muscles cells comes in at F6P and uses hexokinase
fructose in liver cells comes in at GAP and uses glucokinase
is fructose in the liver a substrate for glucokinase?
no, it is a substrate for fructokinase
how does fructose differ in glycolysis in liver cells vs muscle?
in muscle: substrate for hexokinase and phoshorylated to give us F6P
in liver: not a substrate for glucokinase (fructokinase)
what is an issue for fructose coming in at GAP?
the bypass of the PFK-1 mechanism
- not regulated tightly do to bypass of PFK-1 step
-large [fructose] in diet can cause fatty liver
what is needed for fructose to enter the glycolytic pathway? what is it first converted to?
energy (ATP) and the enzyme fructokinase
- converted to fructose-1-phosphate
what is Fructose-1-phospate a signal of? why is fructose-6-phosphate (F6P) not a signal of this?
being in the WELL FED state
-only produced in theliver when there is fructose in the diet
F6P can be coming from gluconeogenesis which would be starvation conditions
what is F-1P converted to using aldoase? where do each of these products go?
Glyceraldehye and DHAP
Glyceraldehyde gets phosphorylated to go into glycolysis
DHAP goes into glycolysis at the isomerizatoin step from GHAP to DAP
what tissue is the entry of fructose into the glycolytic pathway occuring in if fructokinase is being used as an enzyme?
The LIVER
what tissue is the entry of fructose into the glycolytic pathway occuring in if hexokinase is being used as an enzyme?
muscle cells
How is galactose introduced into the glycolytic pathway? what enzyme is needed? at what stage does this occur?
Galactose is introduced as G6P through a series of conversions, beginning with galactokinase (uses ATP)
what sugars can be incorporated into the hexokinase active site?
fructose, mannose and glucose
what types of reactions do aldolases commonly catalyze?
cleavage rxnās
explain stepwise how galactose enters glycolysis
1) Galactose+ATPāGalactose-1-phosphate+UDP-glucose
2) Galactose-1-phosphate+UDP-glucoseāUDP-galactose+Glucose-1-phosphate
- Galactose-1-phosphate is converted to UDP-galactose by exchanging its phosphate group with UDP-glucose
3) G1PāG6P
- G1P is converted to G6P to be used in glycolysis
4) regeneration of UDP-glucose occurs through an empimrase which converts UDP-galactose to UDP-glucose which is needed to continuously supply UDP-glucose for the rxn
what product of the galactose feeder pathway gets shipped into glycolysis?
G1P
-gets converted to G6P
what kind of enzyme is PFK-1? When is it more active vs less?
allosteric enzyme
-more active in the R state and less active in the T state
how many ATP binding sites does PFK-1 have? how does each site differ
2
Substrate site -high affinity for ATP
Allosteric site - binds when [ATP] are high
when is the allosteric binding site of PFK-1 present? what does this do?
Present in the T state, binding of ATP helps stabilize the T state
-decreases affinity
what products increase ATP inhibition?
high [AMP] and [ADP] overcome ATP inhibition and shift binding to the R state
where does F6P bind to in PFK-1 complex?
in the R state
-stabilizes R state
Why is [AMP] a regulator for being in the R state?
AMP, ADP and ATP all compete for the active site, once [AMP] builds up in the cytosol, it will shift the enzyme to the R state
What curve would you expect for the binding of F6P on PFK-1? Why?
sigmoidal because is allosteric
what is the main signal that switches us from T to R state in PFK-1?
Fructose-2,6-BP
what does build up of PEP in the liver indicate?
decreases in pyruvate kinase activity and increases in gluconeogenesis
where does citrate come from? what happens to it when the CAC slows down?
mitochondria (CAC)
-taken out of the mitochondria into the cytosol and gets split into acetyl-coA and Oxaloacetate
-Acetyl coA is then used to make fat
why is citrate a good inhibitor of PFK-1?
When citrate levels are high, it indicates that the cell has a sufficient energy supply or that the citric acid cycle is running efficiently
-when bound to PFK-1 this signals no more energy is needed (rare to have lots of build up)
-rarely builds up in the liver because it is broken down to make fat
why is AMP a more important regulator than ADP?
Its more sensitive
-since it is present in [lower] changes in the [AMP] following the depletion of ATP has a much larger relative change
what are activators of PFK-1?
AMP, ADP and F-2,6-BP
what are inhibitors of PFK-1?
ATP, citrate and PEP
when ADP is not being used as an activator in the PFK-1 rxn, where is sent to and what does it do?
it is sent to the mitochondria to be converted back to ATP in oxidative phosphorylation
why is ADP not a good indicator of the energetic state of the cell?
because ADP can be converted to AMP and ATP by adenylate cyclase
how do low energy (ATP) signals differ in the cytosol vs the mitochondria?
high [ADP] are a signal of low ATP in the mitochondria while high [AMP] are a signal of low ATP in the cytosol
What is a very potent (most important) regulator of PFK-1?
F-2,6-BP
-shifts from the T state to the R state
why is PFK-2 important? where in glycolysis does this happen?
PFK-2 is an important regulator of glycolysis, it is not present in glycolysis itself but regulates the conversion of F6P to F-2,6-BP (activator for the R state of PFK-1)
-produces a major regulator of glycolysis
how does regulation of PFK-2 differ in the liver vs the muscle tissue?
in the liver it is regulated by insulin and glucagon
in skeletal muscle it is regulated by allosteric regulators (not affected by phosphorylation / dephosphorylation)
How does insulin affect PFK-2? why?
insulin will activate PFK-2
-increases F-2,6-BP
-PFK-2 will help stabilize R state on PFK-1 which will drive glycolysis- pulling glucose into the cells
how does glucagon affect PFK-2? why?
glucagon will decrease PFK-2 activity
-decreases F-2,6-BP
-glucagon signals low blood sugar, therefore we do not want to breakdown glucose and would want to slow glycolysis
how does F16BP affect pyruvate kinase?
feed-forward activation of pyruvate kinase
How does Acetyl coA affect pyruvate kinase in the muscle vs the liver? why?
Acetyl coA acts as an inhibitor in the muscle, and has no affect in the liver
-levels of Acetyl coA are unlikely to build up in the liver because it is used to make fatty acids and cholesterol
How does ATP affect pyruvate kinase in the liver vs the muscle?
it acts as an inhibitor in both the muscle and the liver
How does glucagon affect pyruvate kinase in the muscle vs the liver? why?
inhibits PK in the liver and has no effect on PK in the muscle
-no glucagon receptors in the muscle
explain the steps in glucagon binding and activation of protein kinase A:
1) Glucagon binds to its receptor: Glucagon attaches to the glucagon receptor on the plasma membrane of target cells, primarily in the liver.
2) -protein activation: The receptor undergoes a conformational change, activating the Gs protein by promoting the exchange of GDP for GTP on its alpha subunit.
3) Adenylate cyclase activation: The activated Gš alpha subunit binds to and stimulates adenylate cyclase, an enzyme located on the inner surface of the plasma membrane.
4) cAMP production: Adenylate cyclase converts ATP into cyclic AMP (cAMP), increasing cytosolic cAMP levels.
5) PKA activation: cAMP binds to the regulatory subunits of PKA, causing them to release the catalytic subunits, which become active.
6) Phosphorylation of target proteins: The active catalytic subunits of PKA phosphorylate specific enzymes involved in metabolic processes such as glycogen breakdown and gluconeogenesis.
How is PK-L regulated?
by phosphorylation by protein kinase A following the G-coupled binding cascade of glucagon
does phosphorylation activate or inactivate pyruvate kinase in the liver?
inactivate
What is AMPK? what regulates it? why is this important?
AMP-activated protein kinase is a cytosolic enzyme activated by allosteric binding of AMP
-causes phosphorylation which switches cells from sythesis of molecules (ATP consumption) to metabolism (ATP production)
-helps produce ATP when levels are low
what is the difference between AMPK and PKA (cAMP-dependent kinase)?
AMPK responds to energy levels inside the cell while PKA resonds to blood glucose levels outside of the cell
why do cancer cells regulate glycolysis fast that non-cancerous tissue?
they depend on hypoxic (anaerobic) conditions as they do not use oxidative phosphorylation
-HIF-1 stimulates many glycolytic enzymes that bind to promoters of GLUT 1 (high affinity transporters)
how does cancer affect glucose transporters and glycolytic enzymes?
many enzymes and transporters are overexpressed in cancerous tissue
