Three enzymatic reaction are very exergonic, hence, thermodyncamically favorable reaction (Name them). These need to be REGULATED via _______ and ______ _______ mechanisms. Keq is GREATER than ____
Hexokinase (HXK), Phosphofructokinase 1 (PFK1) and Pyruvate kinase (PK) (all three are very exergonic)
allosteric and post-translational mechanisms
What about the REST of the non exergonic reactions? Additionally, what is the range of their ΔG, and what can be said of the rate of their forward reactions?
They are said to be at-equilibrium reactions
Single digit ΔG ranging from -6 to +6kJ/mol. for glycolysis
Rate of forward reaction = rate of reverse reaction
How do you make the thermodynamically unfavorable reactions favorable? (Revisit pg 3 of the module)
a) Le Chatelier's Principle: On one side, you have incredibly high concentrations of molecule A, and on the other side you have an incredibly low concentration of molecule B. The system will always move away from A towards the direction of B, promoting the favor-ability of the reaction "all unfavorable are favorable"
b) "Because they are coupled": Take one unfavorable reaction and another that is VERY favorable, when combined we get a favorable reaction
**never used together always one or the other, waste of energy
State the Keq and ΔG ranges and direction of reaction for the following reactions:
Exergonic reactions: Keq > 1 | ΔG ≤ -10 kJ/mol | to the right
Equilibrium reactions: Keq ≥ 1 | ΔG -6 to +6 kJ/mol | in neither direction
Endergonic reactions: Keq < 1 | ΔG ≥ 10 kJ/mol | to the left
Cells utilize diverse carbohydrate sources. However, ALL sugar molecules must convert to a singular sugar form, glucose. What is the ultimate fate of glucose?
Glucose is ultimately oxidized to yield energy in a series of enzymatic steps
Stage 1 of Glycolysis is the _______ step. What enzyme does the 1st reaction involve? What type of reaction is this?
What is the story and formula?
This is a coupled reaction
Glycogen (broken down in the liver)
Glucose released into the bloodstream
GLUT (Glucose Transporter) helps glucose enter cells
Hexokinase transfers phosphate groups, using ATP, unto glucose
Glucose + ATP → Glucose-6-phosphate + ADP +H+1 (equilibrium lies to the right)
Kinase: an enzyme that catalyzes the transfer of a phosphate group from ATP to a specified molecule.
Each phosphate group attached can be broken to release energy. Name the bond and rank each bond by energy released
Phosphodiester bond energy release
γ (30kJ) > β > α (10kJ)
What is the purpose of the priming step? Why phosphorylate glucose? What is the advantage?
To phosphorylate glucose with hexokinase
Once phosphorylated, glucose is trapped inside the cell
What are the activators and inhibitors of the 1st reaction
Acitivator: ADP, AMP
Inhibitor: Glucose-6-phosphate, ATP (competitive inhibitor)
The six classes of enzymes
The hexokinase (transferase) reaction is a _______ REACTION. Hexokinase phosphorylates the ______ group on the 6th carbon of glucose. Draw Glucose getting phosphorylated
Explain Coupling strategy number 1 for glucose phosphorylation
The structure of (Acyl) CoA is DIFFERENT from ATP and has a tell tale reactive thiol group that will be pivotal in the biosynthesis of coenzyme A (CoA). In ATP hydrolysis we do _______ bond hydrolysis, however, in Acyl bond hydrolysis, we do ________ bond hydrolysis.
phosphodiester bond hydrolysis
THIOESTER bond hydrolysis
Explain coupling strategy #2 Acyl-Coenzyme A (CoA) bond hydrolysis
1) As the Coenzyme A begins to accept an alkyl/acyl group, an acetyl group is formed with a HIGH ENERGY THIOESTER bond
2) The idea is to couple unfavorable reactions with the breaking of about 1 mol of the THIOESTER bonds (Acyl CoA hydrolysis) which releases a lot of energy, making the reaction favorable
**Hydrolysis meaning we have a chemical breakdown of a molecule due to a reaction with water
State an example of the Acyl-coenzyme A bond hydrolysis
The addition of myristoyl or palmitoyl group on to proteins is actually unfavorable
However, upon the hydrolysis of a myristoyl-CoA or palmitoyl-CoA, a lot of energy is released and coupled to protein addition reaction, making it favorable
Not all hexokinase isoforms have the same level of enzymatic behavior. Hexokinase I has a _____ Km value for glucose and is active when there are _____ levels of glucose. Hexokinase IV has a _____ Km value for glucose. It is present in ______ tissue and active ONLY when there are _____ levels of glucose. What is the difference in affinity?
Hexokinase IV has 100 times lower affinity for glucose
Most kinases use ATP as a ______ _______ _____
**Revisit slide 7
phosphate donor cofactor
What is happening here?
Reaction number 2 in Glycolysis
Isomerization of Glucose-6-phosphate into Fructose-6-phosphate
Later cleavage generates two 3-carbon molecules
Glucose-6-phosphate (cyclical) → opens to non-cyclical form → isomerize →cyclical fructose
Reaction 3 involves Phosphofructokinase phosphorylating ________. This is a very _______ reaction. What is the purpose of dual phosphorylation (2-story)? State the activators and inhbitors
NT = Not going to be tested
(1) To generate Fructose-1-6-bisphosphate which can be isomerized into Fructose-2,6-bisphosphate (F-2,6-BP) by PFK-2.
(2) F-2,6-BP increases glycolytic activity by activating PFK-1.
Activators: AMP, ADP and Fructose-2-6-bisphosphate (NT: also Insulin)
Inhibitor: ATP (NT: also High citrate concentration)
Which molecule contains more energy:
As it pertains to the phosphorylation of Fructose-6-phosphate, PFK1 activity is _____ with low intracellular ATP levels, and _____ with high intracelluar ATP levels.
***The sigmoidal curve should have you thinking higher Km and multimeric, while the hyperbolic curve should have you thinking lower Km and monomeric scavenger
_______ function of two mammalian hormones controls glycolytic flux through PFK-2. What is the general function of each domain and which portion is the PFK-2 and which portion is the FBPase-2
The kinase portion adds a phosphate group to a molecule, while a phosphotase removes a phosphate group from a molecule
The 32-250 domain is the PFK-2 portion and the 250-470 domain is the FBPase-2 portion
What are the 2 pancreatic endocrine signaling cells of interest and at which glucose levels do they intervene
α cell aka glucagon: intervenes when blood sugar/glucose levels are too low
β cell aka insulin: intervenes when blood sugar/glucose levels are too high
Glycolysis is ______ active with Fructose-6-phosphate and ______ active with Fructose-2,6-bisphosphate (Why?)
PFK-1 is activated
How do we transition from low blood sugar to higher blood sugar? (5-story)
Pancreatic endocrine signaling directs increase in glucagon concentrations
Glucagon (agonist and peptide hormone) binds to a glucagon receptor, activating Protein Kinase A (PKA)
Protein kinase A consumes an ATP resulting in a phosphorylated (PFK-2)-FBPase-2 complex at its Protein kinase A recognition motif (RRXS/T) (**A kinase-kinase reaction)
This phosphorylation activates the FBPase-2 portion of the complex
FBPase-2 then converts Fructose-2,6-bisphosphate into Fructose-6-phosphate, increasing the activity of gluconeogenesis (making of glucose) and the overall glucose content of the blood
Where is the Protein kinase A recognition motif (state the motif sequence) and which residues are targeted
In the PFK-2 portion of the PFK-2-FBPase-2 complex, there is usually an RRXS/T motif
Most of the time Serine is targeted, however Threonine can be targeted as well
How do we transition from high blood sugar to low blood sugar? (5-story)
Pancreatic endocrine signaling directs increase in insulin concentrations
Insulin (antagoinst) binds to an insulin receptor activating protein phosphotase-1
Protein phosphotase-1 dephosphorylates the PFK-2 on the (PFK-2)-FBPase-2 complex resulting in an active PFK-2
PFK-2 proceeds to phosphorylate Fructose-6-phosphate converting it into Fructose-2,6-bisphosphate
Fructose-2,6-bisphosphate is an allosteric activator of PFK-1 which signals an increase in glycolysis activity and a decrease glucose content in the blood
What exactly does Fructose-2,6-bisphosphate do to the enzymatic behavior of PFK1? How?
Fructose-2,6-bisphosphate activates PFK1
It is a potent positive allosteric effector/regulator of PFK1 activity
F-2,6-BP is an ISOMER of _______, which is a product of _____. Draw Fructose 2,6-bisphosphate
What happens to the product of PFK1 after allosteric activation by F-2,6-BP? Specify how this affects glycolysis
The highly charged F-1,6-BP molecule is opened and cleaved by aldolase and the Glyceraldehyde-3-phosphate (GAP or G3P) can go through glycolysis
This is an overview of the mechanism aldolase uses to cleave F-1,6-BP. What types of catalytic mechanisms are being utilized by aldolase?
Account for: Lys-229 and Asp-33 in both Schiff base formation and hydrolysis
Schiff base formation: Lys-229 is covalent catalysis and Asp-33 is using general base
Schiff hydrolysis: Lys-229 is general base (it picked up a proton) and Asp-33 is general acid
Schiff bases are also called ______
What happens after Aldolase generates GAP and DHAP? Which direction does equilibrium lie in? Why is that direction beneficial? What is the enzyme responsible for making that benefit possible
Isomerization of DHAP to GAP
Equilibrium lies to the left: ~96% DHAP and ~4% GAP
This direction is beneficial because it invokes Le Chatelier's principle, the tendency for larger concentrations of one isomer moving in the direction of the isomer in a smaller concentration
After triosephosphate isomerase moves the equilibrium converts DHAP, how many GAP molecules are present?
Phosphoglucoisomerase converts _______ to _______ while Triosephosphate isomerase converts _______ to _______
aldose to ketose
ketose to aldose
Draw the mechanism:
DHAP to GAP
What happens after we get both GAP molecules? Name the enzyme involved
2 GAP molecules get oxidized by glyceraldehyde-3-dehydrogenase with the help of an inorganic phosphate group and 2 NAD+
The result is 2 molecules of 1,3-Bisphophoglycerate
What does NAD stand for and what is its function? State which of the following is the reduced NADH and which is the oxidized NAD+
NAD: Nicotinamide Adenine Dinucleotide, a cofactor in reduction-oxidation (redox) reactions, in this case GAPDH (DeHydrogenase)
Draw the 5 stage mechanism:
GAPDH (dehydrogenase) takes GAP to 1,3 BPG
What is the role of NAD in GAPDH?
2 NAD molecules, our GAPDH cofactor, carry electrons in form of their hydrogens (1 hydrogen = 1 electron)
When in the mitochondria, the NADHs (mitochondrial) activate complex 1
Complex 1, a mitochondrial protein complex, pumps protons from the mitochondrial matrix to the intermembrane space
The protons are used to generate ATP at a consistent rate, so as a result, each REDUCED NADH is the equivalent of 2.5-3 ATPs
We have 2 NADHs so that means 5-6 ATPs
What are the positive and negative effectors of GAPDH
NADH and ATP reduce the affinity for the cosubstrate
AMP and ADP increase affinity
How are NADH equivalents native to the cytosol, converted into mitochondrial NADH equivalents? (6-story) What is the strategy called?
The Malate-Aspartate Shuttle strategy
NADH can't go across the membrane so Malate dehydrogenase converts Oxaloacetate (reduced) into Malate and the reaction is coupled with NADH getting oxidized into cytosolic NAD+
Malate can go into the matrix but cannot exit, so it activates Oxaloacetate by reducing NAD+ of the matrix to NADH of the matrix
Oxaloacetate is then converted into Aspartate by a transaminase (matrix)
Aspartate is capable of leaving the matrix and enters cytosol
Once in the matrix, aspartate can undergo a reverse reaction with transaminase to generate a cytosolic oxaloacetate which can be converted into cytosolic Malate again by Malate dehydrogenase restarting the process
What happens after our two 1,3 BPG molecules are generated? What is the importance of this reaction?
Both 1,3-BPG with ADP and H+ are converted into two 3-phosphoglycerate molecules and two ATP molecules
The importance is that ATP is finally generated (substrate level phosphorylation)
What are the last 3 reaction after both of our 3-PGs are formed? What is the Net ATP gained?
1) They are converted into 2-PG by phosphoglycerate mutase
2) The 2-PGs are converted into phosphoenolpyruvate by enolase
3) Both phosphoenolpyruvates are converted first into pyruvate (enol form) by pyruvate kinase (cpld with ATP formation) and eventually pyruvate be resonance
4 ATP made - 2 ATP used = 2 ATP
What are the steric consequences of going from 3-PG to 2-PG?
Less steric repulsion to more steric repulsion
What happens to pyruvate after it is generated as the last metabolic intermediate in glycolysis? (4-story)
In Eukaryotes, pyruvate is translocated into the mitochondria using a translocase.
Pyruvate is decarboxylated (removal of CO2) using a multienzyme complex called pyruvate dehydrogenase
After decarboxylation, pyruvate forms acetyl
Acetyl is combined with CoA to form acetyl-CoA
How does regulation of pyruvate kinase get us to a LOW blood glucose level? How can it take us to HIGH blood glucose levels? What are the targets, the transition and results?
Pyruvate kinase is phosphorylated by pyruvate kinase kinase and therefore less active allowing for a low blood glucose levels
Pyruvate kinase is dephosphorylated by Pyruvate kinase phosphotase which makes more active allowing for high blood glucose levels
The targets are serine, threonine and tyrosine and they transition from:
(ROH) → (RO-) → (ROPO32-)
Which results in:
In the pyruvate dehydrogenase complex and its regulation cycle, what are E1, E2, & E3 attached to?
E1 to TPP (thiamine pyrophosphate
E2 to Lipoamide
E3 FAD (oxidized) flavin adenine dinucleotide FADH2 when reduced
In one round of successful PDH complex reaction cycle we generate _____ _______ ______ equivalents
two NADH reducing equivalents
T or F PDH does not go through phosphorylation and dephosphorylation
False PDH goes through both
PDH is _______ when active and ______ when inactive
Name and label the following structures
(cut of single bond goes to H and double bond to O)
Lipoamide is a ________ linked cofactor to a ______ side chain of ____ of PDH