Week 2B: Glycogen metabolism and membrane transporters Flashcards
HC 11-14
Structure free glucose
Contains a very polarized reactive aldehyde group
/H
-C (delta plus) = O (delta minus)
The aldehyde group can react with (in vitro, in vivo, intramolecular)
In vitro: Fehlings reagent (Cu2+ ions)
In vivo: amino groups of proteins
Intramolecular: with the C5 hydroxyl group (count from aldehyde, so opposite one) > form ring
Glucose is a …. sugar
reducing
> can reduce Cu2+ to Cu+ in vitro
> aldehyde group is oxidized to carboxyl group (C(=O)-OH) > gluconic acid
> reducing sugars need free reducing ends
Fehlings test for reducing sugars
Solution of Cu2+ (Fehlings reagent) used in vitro to distinguish reducing and nonreducing sugars.
> glucose and fructose are reducing sugars
> Reducing Cu2+ to Cu+
> 2 Cu+ react with water to form Cu2O (red precipate, red/brown color)
A reducing sugar can reduce a …
another molecule while being oxidized themselves from a sugar with an aldehyde group to a carboxyl group
Two ways of adding glucose to proteins
-Glycosylation: enzymatic transfer of glucose in condensation reaction
-Glycation: nonenzymatic spontaneous and irreversible transfer of glucose
Glycation in vivo
Takes place in vivo between glucose and free amino groups of proteins, such as abundant proteins serum albumin and hemoglobin.
> formation of reversible Schiff base
> conversion to stable ketoamine (irreversible)
Glycation of hemoglobin
-Aldehyde group of glucose reacts with N-terminus of the N-terminal Valine residue of HbA (spontaneous reversible)
> formation Schiff base: C(-H)=N-Val-HbA
> Amadori rearrangement (irreversible, spontaneous)
> HbA1c: C1 of glucose like this: C(=O)-H2C(nr1)-N(H)-Val-HbA
Schiff base bond
-C=N-
How is it possible that the amino terminus of HbA beta chain (tetramer with two alpha and beta chains) is valine and not methionine
Hydrolysis of the N-terminal residue
Glycation of HbA at the..
alpha-amino group of this amino-terminal valine residue of HbA beta chain
> in lesser extent at amino groups at lysine side chains in alpha and beta chains of hemoglobin
Does the glycation of HbA to HbA1c impact the function of hemoglobin
no
The rate of glycation of hemoglobin is proportional to…
blood glucose concentration
Normal HbA1c/HbA% level
5.5%, when hyperglycemia, this can rise
The glycation of stable colagen (ECM protein) increases with …
age
> resembles Maillard reaction in food cooking
The aldehyde group of glucose can react intramolecularly with the c5 hydroxyl group. In which ways
-alpha glucose: c1 hydroxyl group faces away of the oxygen in the chain (36% prevalence)
-beta-glucose: hydroxyl group on C1 faces towards the O (from the C5 hydroxyl group) in the ring
> the cyclic alpha and beta anomers (different conformations of the aldehyde group C1 of glucose)
The conformation of the hydroxyl group on C1 on the cyclic glucose isoforms are called:
Alpha- Axial
Beta- Equatorial, in line with molecule (horizontal)
Glycosylation of glucoses
During enzym-catalyzed reaction, cyclic glucose forms stable covalent glycosidic bond via its C1 anomeric position
Glycosidic bond
reducing end Glucose C1 - O - C4 glucose nonreducing end
After glycosylation, the intrachain glucose cannot ..
adopt an open-chain form anymore
How is a linear homopolymer of glucose residues called?
Glucan
In a glucan, a free reducing end can adopt an open chain formation and keep reducing. In an alpha-1,4-glucan. What is the reducing end?
The end with the free C1 carbon (aldehyde group, reactive)
Initiation formation of proteo-glycogen
Start with glycogenin (GN), the initiator/primer.
> GN is a homodimer and both monomers can glycosylate each other
> each GN monomer is glycosylated specifically on a single tyrosine residue (with the benzene ring with OH group, which forms a bond with the reducing (C1) end of glucose).
> Auto-glycosylation
How long does auto-glycosylation of the GN monomers take place?
Until maximally 8 glucose residues added per glycogenin
> monomers dissociate
Elongation glucose chain on glycogenin
By enzyme glycogen synthase (GS)
> GS is a processive enzyme
> Extends existing chains
Branchin in proteo-glycogen
Branchin enzyme (BE) removes an oligosaccaride of 7 glucose residues from the nonreducing end (within the growing chain, 7 glucoses from the reducing, growing end)
> transfer the oligosaccharide to create a new branch point which is at least 4 glucose residues away from a preexisting branch point upstream.
Growth glycogen idea model of Whelan
Growth by glycogen synthase and branching enzyme: forming shells
> Shells: layers of glucose chains created by new branching. Count the branch points from start to end > three branch point till reaching end: four shells (model of Whelan)
Mature glycogen is sphere with about .. shells. It consists of linear units of …-glucose residues. Each glycogen unit has averagely .. branch points connected through … bonds
tmax= 12, g-c=13, r=2
> 12 shells maximally
> linear units of 13 alpha-glucose, connected through (1-6)-alpha-glycosidic bonds
> 2 branch points per glycogen unit on average
The nonreducing ends of glycogen are located at …
the termini of the outer most shell, forming the surface of the glycogen molecule
> excellent acces for glycogen modifying enzymes
> Glycogenin enzyme located in centre of proteoglycogen
Where are glycogen granules localized?
in the cytosol
How is glycogen metabolism regulated in the liver and muscle?
-Liver: glycogen metabolism is regulated to maintain the blood glucose level
-Muscle: glycogen metabolism is used to maintain its own energy requirements (lacks G6Pase) (much glycogen from subsarcolemma glycogen granules compared to myofibrilar glycogen granules before exercise.)
HC12: Glycogen synthase catalyzes …. by using … as a substrate
addition of glucose residues (alpha) at the nonreducing end of growing glycogen chain.
Substrates: [glucose]n (growing glycogen) + UDP-glucose > [glucose]n+1 + UDP
Structure UDP-glucose
Activated form of glucose (like acetyl-CoA is activated form of acetate) > contains high energy phosphoanhydride bond (1) which favors the reaction
UDP-glucose synthesis
Glucose 1-phosphate + UTP > UDP-glucose
Linkages between glucose residues in linear chain glycogen and at a branch point
In linear chain: alpha-1,4-glycosidic bond
In branch point: alpha-1,6-glycosidic bond
How many ATP costs the linkage of one glucose from blood to glycogen
2 ATP, one to regenerate UTP from UDP, and one to activate glucose to glucose-1-P via G-6-P.
Cleavage of a bond by addition of Pi
Phosphorolysis
How is a glucose residue removed from glycogen at the nonreducing end
Adding a phosphate to the reducing end to free it
> phosphorolysis by Glycogen phosphorylase (GP) > needs Pi as cosubstrate
- [glucose]n+1 + Pi > [glucose]n + G-1-P
Debranching enzyme of glycogen
By debranching enzyme
> bifunctional enzyme with transferase activity and alpha-1,6-glycosidase activity
Debranchin glycogen
-Phosphorylase cleaves all glucose until the original chain and branch have 4 glucose left until branch glucose unit. (using Pi, release glucose as G-1-P)
-Transferase (only works when 4 glucose left till branch linkage) remodels so that the outer three glucose units from the branch chain are transferred to the original chain
-a-1,6-glucosidase uses H2O to remove last glucose unit of branch
-phosphorylase can cleave bonds until next branch
How is the glucose cleaved by a-1,6-glucosidase during glyccogenolysis released?
As free glucose
> glucose units at branch points
> free glucose are phosphorylated by glucokinase (liver) or hexokinase (muscle)
> costs extra ATP.
> 1 in 10 glucose residues
Glycogenolysis components
1: release of G-1-P
2: remodeling of glycogen
3: conversion of G-1-P to G-6-P (by phosphoglucomutase)
4: production of free glucose (liver and kidney) from G-6-P
Fates G-6-P
-Glycolysis, in muscle, brain or red blood cells (example)
-Conversion to glucose in liver or kidney (for other organs)
-PPP
Conversion by phosphoglucomutase
G-1-P binds the phosphorylated serine’s phosphate group at C6
> formation intermediate: glucose-1,6-bisphosphate
> Serine of the enzyme binds phosphate group of the C1 carbond
> glucose-6-P as product
Glucose producers and consumers
Producers: liver and kidneys (contain glucose-6-phosphatase)
-Consumers: brain, adipocytes, erythrocytes, muscle cells
Different rates of glycogen breakdown in different exercise
Highest rate in highest intensity (150% of teh intensity at VO2max maximal oxygen consumption)
When glycogen use in fasted state?
During first day (short term response, <12 hrs)
HC13: which type of glucose synthesis mechanism is used early in the morning
Gluconeogenesis (after long period withour meal)
Regulation types in glycogen metabolism
-Hormones (control from outside the cell: insulin, glucagon, adrenaline): (de)phosphorylation
-Metabolites (AMP/ATP, G-6-P, glucose: control from within the cell): allosteric regulation
Epinephrine origin
It is a catecholamine, derived from the amino acid tyrosine (ring with OH in structure) (but contains no amino acids)
> promotes glycogen breakdown
> fight or flight
Structure of glycogen phosphorylase
-Homodimer
Prosthetic group GP
Pyridoxal phosphate (PLP) group bound via Schiff base linkage (aldehyde to amino bond with -HC=NH-) to a lysine residue (epsilon amino group)
PLP is vitamin?
Vitamin B6
PLP function in GP
> contributes to binding of inorganic phosphate for phosphorolysis.
is more energy efficient than breakdown of glucose and phosphorylation by Hexokinase using ATP.
PLP group with its phosphate crucial to bring phosphate into position for catalysis
Glucagon has an important … residue
N-terminal Histidine, important in activation mechanism of the receptor
Isoforms of glycogen phosphorylase
phosphorylase-a: phosphorylated: often active (in R-state)
phosphorylase-b: dephosphorylated: often less active (in T-state)
How does phosphorylation of GP induce active enzyme
water shell is formed around the phosphate: conformational change.
R-state and T-state jargon also applies to…
hemoglobin.
Can the a and b formd of GP exist in both the relaxed and less active tense state?
Yes, but mostly b in T and a in R –> but allosteric regulation overrules the phosphorylation/hormonal regulation
When does GP-b change to GP-a
From fed to fasted state
How many encoded GP enzymes?
Two isozymes
-Muscle GP
-Liver GP
> mostly similar
> different (metabolite) regulations
Allosteric regulation liver GP
The liver GP-a form undergoed R>T transition when glucose binds, inactivating it and overruling the phosphorylation state at the two tails of the homodimer.
Allosteric regulation of muscle GP
At low energy (high AMP/ATP), favors the transition to R-state.
> high concentrations of ATP or G-6-P: shift muscle-GP to T-state
Hormonal regulation liver GP and muscle GP
-Liver GP
> activation by glucagon and adrenaline
-Muscle GP
> activation by adrenaline and calcium (nerve excitation)
> Muscle has no glucagon receptors.
ATP can bind to … of GP in the … isozyme
Nucleotide binding sites in the muscle isozyme
Is glycolysis upregulated by glucagon in the muscle?
No, it has no glucagon receptors
> does react to adrenaline
effect epinephrine to liver lactate which came in from muscle
Gluconeogenesis in fasted state. (glucagon rules)
How does glucagon initiate activation of GP and inactivation of GS (reciprocal regulation)
Glucagon signaling: PKA active.
PKA phosphorylates GS: to T state
PKA phosphorylates glycogen phosphorylase kinase (activation)
GP kinase phosphorylates and activated GP
Why extra step throug GP-kinase
Extra layer of regulation
> phosphorylase kinase can be only be activated by Ca2+ in the muscle (excitation)
> GP-kinase has delta subunits: Ca2+ sensor calmodulin: binds Ca2+ > alpha and beta subunits can be phosphorylated by PKA.
Regulation glycogen synthase by PP1
PP1: protein phosphatase 1(dephosphorylation)
> PP1 inactivates phosphorylase kinase and therefore GP
> PP1 activates GS
> PP1 dephosphorylates GP directly as well
Regulation by PP1 in muscle and structures/interaction
Catalytic subunit of PP1 is active while bound to regulatory subunit Gmuscle.
> PKA phosphorylates Gmuscle, PP1 released, becomes less active
> PKA phosphorylates and activates an inhibitor of PP1 as well
> result: GP and GS can shift to phosphorylated form and GP active and GS inactive
Regulation PP1 in liver
Catalytic subunit PP1 bound to regulatory subunit Gliver
> glucose brings GP in T-state, allowing PP1 to dephosphorylate and inactivate GP: glucose is bound to Gliver, Gliver releases. Free PP1 is active in the liver (Gliver is inhibitor).
>Free PP1 inactivates GP but activates GS
Effect insulin on glycogen metabolism
Insulin binds and activates intracellular protein kinases like PKB/Akt
> kinases phosphorylate and inactivate glycogen synthase kinase, thereby inactivating kinase activity (no inhibition GS).
> this allows PP1 to dephosphorylate and activate GS
How much ATP costs GP-b > GP-a
2 ATP, 2 times phosphorylation.
Infusion of blood glucose in blood stream leads to this transition in glycogen metabolism enzymes
-Inactivation of GP first, followed by activation of GS.
> activation GS through insulin (longer)
> inactivation of GP by glucose allosterically: directly
Glycogen storage diseases: defect in Von Gierke
Defect G6Pase
> liver and kidneys
Pompe disease defects
a-1,4-glucosidase (and therefore glycogen breakdown)
> all organs
> part of the debranching enzyme
Andersen disease enzyme defect
Defect branching enzyme
> liver and spleen
McArdle disease defect
Glycogen phosphorylase
> in muscle: only muscle isozyme affected
McArdle disease complication
strenuous exercise cause painful cramps, reversible: relief after a while
> no glycogen breakdown, no release of myosin in muscle
> muscle goes over to other reserves: you ok again: ATP regeneration by exercise: anaerobic and aerobic metabolism.
Cellular symptom Pompe disease
Lysosomes full of glycogen
> no breakdown
> only little breakdown percentage in lysosomes, most in cytosol but this is defect in Pompe disease.
HC14: Why were membrane proteins hard to study?
-Hard to crystallize
-We cannot predict 3D structures from amino acid sequence
Revolutions in protein studying
-Alphafold predictions
-Cryo-EM (crystallizing not needed)
Driving force and transport mechanism to enter enterocytes, example Ca2+
No driving force (dG=0) > no net transport
> electrochemical gradient
> Ca2+ is positive, charge membrane is -70mV
dG and dribing force
dGinward = RTln(Cin/Cout) + ZFdV
Z= charge molecule
>
dG < 0: driving force for transport
dG=0: equilibrium
dG > 0: requires active transport, simply add dG of coupled reaction
What is a drug is not charged. When is active transport needed?
When the concentration in plasma is lower than intracellular.
dGinward > 0
> 310K as body temperature (37 K + 273 K)
> dV is membrane potential -70 mV > -70/1000 V
> F and R are constants
is an equilibrium reached for massive fluxes across epithelia
No, driving force for transport remains
Characteristics channels and transporters
-Both regulated, high variation in max transporters, several mix-forms found.
If Na+ is replaced with a neutral molecule and drug transport is abolished then …
probably cotransport drug and Na+
Channel activity measured with
Patch-clamp measurement
Types of transporters
-Facilitated diffusion (passive)
-Symporters
-Antiporters
-ATP-hydrolysis driven.
Properties of transporters
-Saturable
-Inhibitable
-Temperature sensitive
Methods in transporter research
-Transwell plate
-Inside out vesicles (extracellular leaflet switched to inside)
Difference carrier mediated transport and simple diffusion on kinetics
Carrier mediate: hyperbolic
Diffusion: linear
Rate of transport
Uptake per time unit
About … % of human genes are transporter related and about … % of them are transporters/channels themselves
10%, 50%
SLC superfamily
Solute Carrier (400 members)
> SLCs do not directly rely on ATP hydrolysis
ABC transporters
(49 genes)
ATP binding cassette transporters family
> rely on ATP hydrolysis
1: bind substrate
2: bind ATP: conformational change and release substrate
3: hydrolysis to ADP and Pi
4: release and reset
P-type ATPases
Covalent phosphorylation of catalytic aspartate
> ATP dependent
> transport, Na+/K+, H+, Ca2+, Mg2+, phospholipids, heavy metals
What happens to Abcg2-/- mice when fed diet containing pheophorbide a?
Ear lesions
> fungal breakdown product of chlorophyll: pheophorbide a
> fermened batch of alfalfa
Abcg2 function
Secretion product into the bile (for excretion) by the liver or from the portal vein directly back.
Name an anticancer drug affected by membrane transporters?
Abcb11 > resistance against ivermectin
What if defect Abcb11?
Dose of ivermectin against cancer should drop, less resistance against the drug
Drug-drug interactions and transporters example cerivastatin and gemprifozil
Gemprifozil inhibits OATP1B1 > less statin metabolism
> increase plasma concentration of cerivastatin > lethal
Why can anti-cancer drugs by less effective
-Activation DNA repair genes
-Blocked apoptosis
-Activation of detoxifying systems (CYP)
-Decreased influx
-Increased efflux by ATP dependent efflux pumps.
What is NTCP
A symporter which is present in only the liver and facilitates cotransport of sodium with bile acids
NTCP functions (influx bile acids)
-Bind bile acid receptors like TGR5: >Glucagon-like peptide 1 secretion > improved glucose metabolism
> Less inflammation
> increased energy expenditure via brown adipose tissue.
-Bile acids bind FXR in nucleus:
> less inflammation, less fibrosis, less gluconeogenesis, reduced FXR signaling in NAFLD.
How to inhibit NTCP
Gene therapy with virus HBV which can use NTCP as docking receptor ,
or Myrcludex B
NTCP KO leads to … conjugated bile acid
much more
NTCP inhibition by Myrcludex B induces … i obese mice
body weight loss in obese mice
What happens to bile acids in blood when NTCP is downregulated
> it stays longer in the blood after meal and later a decline
Peptidic NTCP: NTCPi has a clinical application. Result inhibition NTCPi
reduced
-obesity
-steatosis
-atherosclerosis
-inflammation
