1
Q
When does the pancreas release insulin?
A
When blood glucose levels are > 5.5mM
2
Q
What happens in the pancreas when blood glucose is < 5.5mM?
A
- some ATP is generated but not enough to inhibit Katp channels
- membrane stays hyperpolarized and Ca2+ channels stay closed
- no insulin leaves the B cells
3
Q
What happens in the pancreas when blood glucose is > 5.5mM?
A
- lots of ATP is generated and this inhibits Katp channels
- membrane becomes depolarized causing Ca2+ channels to open
- insulin leaves the B cell and enters the blood stream to stimulate glucose uptake
4
Q
What glucose channels are in the pancreas?
A
GLUT 2 –> insulin independent channels (passive diffusion)
5
Q
Where is GLUT 4 found and what is it?
A
muscle and adipose tissue ONLY
- glucose channels that get inserted into the membrane when the cell is stimulated by insulin (still passive diffusion)
6
Q
How does glucose from the diet enter the liver?
A
PORTAL VEIN
7
Q
How does glucose enter the liver?
A
GLUT 2 channels (insulin independent)
8
Q
What is the first thing that happens to glucose when it enters the liver?
A
HEXOKINASE and GLUCOKINASE phosphorylate it to become G6P to sequester it inside the cell
9
Q
What are the 3 pathways that G6P can undergo once in the liver?
A
1) GLYCOLYSIS –> to form pyruvate and then acetyl coA
2) GLYCOGEN SYNTHESIS –> to store (when fed)
3) PENTOPHOSPHATE PATHWAY
–> precursor for membrane lipids and nucleotides
10
Q
How does glucose enter into muscle cells?
A
GLUT 4 channels
11
Q
What happens to glucose when it enters the muscle cell?
A
HEXOKINASE converts it to G6P
12
Q
What can happen to G6P once in the muscle cell?
A
1) GLYCOLYSIS
2) GLYCOGEN SYNTHESIS
13
Q
What is the difference in glycogen pool in the liver and muscle?
A
95% in liver and 5% in muscle
14
Q
What is the difference in glucose fate in the FED or FASTING/EXERCISING state?
A
FED –> glucose is stored as glycogen via glycogen synthesis
FASTING –> glucose is broken down vis glycolysis to pyruvate -> acetyl coA -> NADH -> ATP
15
Q
What channels uptake glucose in fat cells (adipocytes)?
A
GLUT 4
16
Q
What happens to glucose when it enters fat cells?
A
HEXOKINASE phosphorylates glucose to G6P and this then goes to acetyl coA –> oxphos or acc fas
17
Q
In the fasting state what happens in the fat cells?
A
FA is supplied for energy, not glucose (b/c no glycogen stores here)
18
Q
What kind of channels are in the kidney? What happens to glucose when it enters?
A
- GLUT 2 channels
- glycolysis ONLY, right away
19
Q
What kind of channels are in the brain? What happens to glucose when it enters?
A
- GLUT 3 channels
- glycolysis right away and energy used for the brain
- fully dependent on glucose from circulation because no storage
20
Q
What are the 4 stages of energy expenditure?
A
1) ATP stored in the muscle is used first
2) ATP produced by Creatine Phosphate and ADP
3) glycogen stores in muscle are broken down to provide glucose to be oxidized (anaerobic)
4) ATP is generated by breakdown of glycogen and lipids by aerobic pathways
21
Q
What inhibits and promotes glycolysis?
A
(-) –> high ATP, low O2 or low NADH/FADH2
(+) –> low ATP
22
Q
What is produced by the CAC?
A
NADH, FADH2 and CO2
23
Q
What inhibits the CAC?
A
(-) –> high NADH
24
Q
What is produced by oxidative phosphorylation?
A
32 ATP
25
What inhibits and promotes ox phos.?
(-) --> low NAD+, high ATP
(+) --> low ATP
26
What is the main difference between HEXO- and GLUCO- kinase?
Hexokinase is quickly saturated (high Km) and Glucokinase has a lower Km value so it works when hexo- is saturated in liver cells only
27
What is THE COMMITTING STEP of glycolysis and why?
Step 3: PFK
- committing because it takes F6P and irreversibly makes if F16BP, meaning that F6P can not longer go back to G6P and follow a different pathway
28
What activated PFK and what inhibits it?
(+) --> AMP, F26Bp
(-) --> ATP, citrate, lactate
29
What are the two states of PFK?
R state --> active
T state --> inactive
30
Which is used in glycolysis: DHAP or GAP?
GAP! That is why DHAP>>GAP in cells because GAP is quickly siphoned off by glycolysis
31
What is special about 13BPG and how does this help promote glycolysis?
It is a high energy intermediate so therefore the next step will occur quickly (highly favoured) to get rid of the high energy intermediate
- SUBSTRATE CHANNELING
32
What is special about PEP?
A high energy intermediate so pushes the reaction towards pyruvate formation
33
What activates and inhibits pyruvate kinase?
(+) --> F16BP
(-) --> high ATP
34
What is the difference in ATP produced in anaerobic vs aerobic glycolysis?
anaerobic --> 2 ATP
aerobic --> 32 ATP
35
What is the CORI CYCLE and when does that happen?
--> liver produces glucose from pyruvate (from lactate) and sends it to muscle for the muscle to use and convert to pyruvate
to lactate and back into liver
--> pyruvate<->lactate catalyzed by LDH
--> happens in anaerobic conditions
36
Where are MCT1 and MCT4 located and what do they do?
MCT1 --> in liver
MCT4 --> on muscle
- both transport lactate (MCT1 bring it in and MCT4 brings it out)
37
How does the muscle convert glucose to pyruvate in anaerobic conditions?
HOMOLACTIC FERMENTATION
38
Why is lactate exported to blood from muscle after homolactic fermentation?
low pH (high lactic acid concentration) inhibits PFK1 activity which would prevent the breakdown of glucose
39
When is the WARBURG effect seen and what is it?
CANCER CELLS: ferment glucose to lactate even in presence of O2... use mainly glycolysis as a source of ATP (get ~4 ATP vs 32) because it is quick
40
What is GLUCONEOGENESIS?
producing glucose after glycogen is completely exhausted (from non-carb sources)
41
What is gluconeogenic pathways in the liver and where does the energy for this come from?
1) LACTATE --> pyruvate
2) Pyruvate --> G6P
3) G6P --> glucose
- the E comes from the hydrolysis of triglycerides
42
What is stimulated by low blood glucose?
GLUCAGON release from alpha pancreatic cells
43
What is the starting point of gluconeogenesis?
OXALOACETATE
44
Where does GLUCONEOGENESIS occur?
Mainly in the LIVER and a small bit in the kidney
45
What are the first and second irreversible step of gluconeogesis?
PYRUVATE --> oxaloacetate
- activated by acetyl coA
- uses an ATP
OXALOACETATE --> PEP
- substrate channeling
- uses a GTP
46
What is the third irreversible step of gluconeogenesis?
F-1,6-BP --> F6P
- removes a phosphate
- inhibited by F26BP and AMP
47
What is the fourth irreversible step of gluconeogenesis?
G6P --> glucose
- "frees" glucose from the liver to feed tissue
48
What is unique about the last step of gluconeogenesis?
G6Pase is ONLY IN THE LIVER and is regulated at the transcriptional level (increased transcription by glucagon)
49
If the pancreas senses LOW BLOOD GLUCOSE what will happen?
GLUCAGON released by alpha cells of pancreas leading to CATABOLIC reactions (use energy to break down molecules to produce glucose)
- glycogen breakdown
- lipolysis (for E)
- gluconeogenesis
50
If the pancreas senses HIGH BLOOD GLUCOSE what will happen?
INSULIN released by beta cells of pancreas leading to ANABOLIC reactions (accumulating energy to generate storage molecules for glucose)
- glucose UPTAKE (GLUT 4 insertion)
- glycogen synthesis (increased glycogen synthase in hepatocytes)
- lipogenesis
51
What is the function of ADK?
Restores ATP from 2 ADP, also produces an AMP (activates PFK)
52
Is ATP or AMP a stronger regulator of PFK?
AMP is! Even though in lower concentrations it overcomes the ATP inhibition and activates PFK
53
With low blood glucose in the liver what is favoured?
GLUCONEOGENESIS due to activation of FBPase 2 and inactivation of PFK2 (DECREASE IN F26BP)
54
With stress what happens in the heart in terms of glucose use?
GLYCOLYSIS: due to PFK2 activation and F26BP increasing in concentration (inhibits FBPase2)
55
Where does most fructose metabolism occur and what enzyme two enzymes are unique to the liver?
in the LIVER (95%) where fructokinase and F1P are used (aldolase B)
56
What is special about FRUCTOKINASE?
It isn't allosterically inhibited like hexokinase
57
What are the two options for fructose metabolism?
1) MAIN PATH: GAP produced from glyceraldehyde and this is moved straight into glycolysis
2) IF CELL NEEDS LIPIDS: produces Glycerol-3-phosphate which can be used for TGs and can also continue to DHAP which can be used to make GAP for glycolysis as well
58
What two enzyme does the muscle use for fructose metabolism that are different from the liver?
hexokinase for step 1
F1,6P (aldolase A) for step 2 to produce GAP and DHAP
59
How much fructose metabolism is done in the muscle?
5%
60
Where does GALACTOSe enter glycolysis?
G6P
61
Where do fructose (muscle) and mannose enter the glycolysis?
F6P (before PFK control), so could still be used elsewhere
62
Where does fructose (liver) enter glycolysis?
GAP (after PFK control)
63
What type of organ is the liver in FED state regarding GLUCOSE?
ANABOLIC: generates glycogen
- this happens ahead of the PFK control, so unregulated
64
What type of organ is the liver in FED state regarding FRUCTOSE?
ANABOLIC: generates FAs, because after PFK control so cannot go backwards
65
What is the LELOIR pathway?
Galactose metabolism
- mostly in the liver
- undergoes step 1 by galactokinase (NOT HEXOKINASE)
- uses UDP-galactose to replenish UDP glucose allowing G1P to be created
- G1P can then be converted to G6P to be used for glycolysis
66
Where is MANNOSE metabolism found and what is the first enzyme?
In glycoproteins
- hexokinase catalyzes the first step
67
What happens in TYPE 1 DIABETES?
- autoimmune destruction of insulin-producing B cells in pancreas
- no insulin secretion
(INSULIN DEPENDENT DIABETES)
68
What happens in TYPE 2 DIABETES?
- insulin "resistance", loss of sensitivity (no GLUT 4 recruitment)
- initially B cells over produce insulin to compensate, but then they get exhausted and low insulin is produced to mimic T1
(INSULIN INDEPENDENT DIABETES)
69
What happens with HYPERGLYCEMIA?
- glycation of proteins (chemical addition of simple sugars to circulating proteins)
- uncontrolled process, damages proteins
(NOT GLYCOSYLATION: ER control, complex sugars added to folding protein)
70
What is FRUCTOSEMIA?
hereditary fructose intolerance
- either fructokinase mutation or F16BP mutation (more severe b/c fructose already trapped in cell)
71
What is GALACTOSEMIA?
- any one of the 3 main enzymes can be mutated
- rare autosomal recessive genetic disorder
- milk is TOXIC to newborns with this condition
72
Can a galactosemic mother still produce milk?
YES! She can generate UDP-galactose from UDP-glucose using energy from UDP-glucose formation (inverts epimerase reaction)
73
What is the purpose fo the PPP?
to generate nucleotides, phospholipids, and cholesterol for dividing cells (intermediates for cell division)
74
Where does the PPP branch out and branch back in to glycolysis?
OUT: G6P
IN: F6P and GAP
75
What is the RLS of the PPP?
G6PDH: generates RuBp from G6P and releases NADPH
76
Where does the PPP mostly occur?
LIVER (~30% of G6P goes here)
- adipose tissue
- RBCs
77
What is NADPH used for?
FA synthesis and cholesterol synthesis
78
What is Ru5P a precursor for?
ATP, GTP, TTP, CTP
79
Compare the ratios of NAD+/NADH and NADP+/NADPH.
NAD+/NADH = 1000
- much more NAD+ in the cell because this drives glycolysis forward
NADP+/NADPH = 0.01
- much more NADPH because needed for synthesis of things!
80
How many G6P does the PPP start with?
3
81
What are the 3 steps of the PPP?
1) oxidative
2) isomerase/epimerase
3) recycling reactions
82
What is the purpose of the OXIDATIVE phase of the PPP?
- to generate NADPH
- the ONLY irreversible step of PPP
- highly specific to NADP+
83
What is the purpose of the ISOMERIZATION/EPIMERIZATION phase of PPP?
- to generate R5P
84
What is the purpose of the RECYCLING REACTIONS of PPP?
- to recycle R5P and Xu5P to generate 2F6P and GAP for glycolysis
85
What regulates PFK and G6PDH in terms of deciding the fate of G6P?
PFK: inhibited by ATP
- ATP prevents glycolysis and pushes G6P to PPP
G6PDH: inhibited by NADPH
- NADPH prevents PPP and pushes G6P to glycolysis
86
If cell needs NADPH and nucleotides what happens in PPP?
- NADPH and R5P are both produced
- excess R5P and Xu5P are recycled back into glycolysis as F6P and GAP
87
If cell needs only NADPH (not nts) what happens in PPP?
- NADPH and R5P are both produced
- all R5P and Xu5P are recycled to glycolysis
88
If cell only needs nucleotides what happens in PPP?
- reaction won't go forward because NADPH will inhibit G6PDPH
-F6P and GAP can be used for reverse synthesis of R5P for nucleotide synthesis
89
What effect do cancer cells have on the PPP?
- up-regulate it so even more glucose is used up
- increases the transcription of G6PDH (RLS)
90
What is an alternative benefit of NADPH??
- maintains GSH and prevents hemolysis
91
What is GSH and how does it prevent damage to RBCs?
- GSH binds ROOH compounds (produced by RBS --> H2O2) and sequesters them
- without GSH (without NADPH), these oxidants damage cell membrane and cause cell lysis
92
What cycles happen with O2 vs without O2?
WITH O2 --> glycolysis and ox. phos. to produce NAD+ and sustain glycolysis
WITHOUT O2 --> cori cycle in liver to regenerate glucose from lactate
93
Why is glucose converted to glycolysis?
It doesn't disturb osmotic pressure as would an equal amount of glucose (~50,000)
94
What does the breakdown of glycogen produce?
G1P
95
What happens to glycogen that is broken down in muscle?
Mostly used to produce energy for contraction, as it doesn't have G6P phosphatase so cannot export G6P as glucose to blood
96
What happens when glycogen is broken down in the liver?
Used to maintain blood glucose levels
- selective G6P expression in liver which allow G6P to be exported as glucose into the blood vis T2
97
Where is G6P phosphatase located?
in the ER membrane
98
What are the reducing and non-reducing ends of glycogen?
REDUCING: one reducing end and this is where glycogen forms from (bound to glycogenin)
NON-REDUCING: many non-r. ends and this is where new glucose is added on
99
What bonds are formed between adjacent glucose molecules in glycogen? (normal and at branch points)
normal --> a(1-->4) linkage
branch pt --> a(1-->6) linkage
100
What enzymes promote glycogen synthesis and breakdown and where are they located?
SYNTHESIS:
-UDP glucose phosphorylate
- glycogen synthase (RLS)
BREAKDOWN:
- glycogen phosphorylase
These enzymes are located on glycogen itself
101
What makes the synthesis of UDP-glucose from G1P irreversible?
- coupled to inorganic phosphatase activity (use of PPi)
102
What is GLYCOGENIN?
- primes synthesis of a new glucose molecule
- one per glucose, contains glycosyltransferase to add an 8 glucose primer
- bound by reducing end of glycogen
103
What is GLYCOGEN SYNTHASE?
- enzyme physically bound to glycogenin
- elongates a pre-existing glycogen chain up to a point (b/c bound to origin)
- releases UDP when glucose is added onto the chain by a(1-4) linkage
104
How is UTP regenerated after UDP is released from glycogen synthase?
UDP + ATP <--> UTP + ADP
- undergone by nucleotide diphosphate kinase
105
How are branches generated in glycogen and what are the rules for this?
BRANCHING ENZYME
- addes new a(1-6) glucosyl branch points by moving chains
- transfers ~7 glucosyl residues to the C6-OH
- each transferred segment must come from a chain of 11 residues minimum
- new branch point must be >4 residues away from other branch points
106
What is the glycogen balance sheet for one glucose addition?
Glucose + 2ATP + g(n) + H2O --> g(n+1) + 2ADP + 2Pi
107
What are the 3 steps of glycogen breakdown?
1) generation of G1P
2) debranching
3) conversion of G1P-->G6P
108
How are glycosidic bonds cleaved?
PHOSPHOROLYSIS: adding a P to glucose as it is removed to produce G1P
(hydrolysis on the other hand produces a glucose)
109
What makes the glycosidic bond cleavage favourable?
- although dG' = 3.1 kJ/mol, the intracellular Pi concentration is so much greater than G1P, so
110
When and how does the debranching enzyme work?
When only 4 glucose residues are left in a branch, GLUCOSYLTRANSFERASE
works by moving the distal 3 to another branch at the a(1-->4) bond so they can be phosphorylated
- the last glucose at the branch a(1-->6) linkage will be hydrolyzed by a(1-->6) GLUCOSIDASE and turned into glucose
111
What percent of glycogen becomes G1P? What does the rest become?
92% --> G1P
8% --> glucose
112
What happens in muscle vs liver when glycogen is broken down?
MUSCLE: G6P continues into glycolysis to generate ATP for muscle contraction
LIVER: G6P converted to glucose and goes into circulation
113
What converts G1P to G6P?
Phosphoglucomutase
114
What is the ATP produced/used by glycogen synthesis or breakdown?
SYNTHESIS: 2 ATP consumed per glucose
BREAKDOWN: 33 ATP produced per glucose (33 because breakdown gives G6P so saving an extra ATP)
OVERALL = 33-2 = 31 (97%)
115
What is IRREVERSIBLE covalent protein modification?
ex: protease activity
- unable to get back to starting material
-cutting molecule smaller
116
What is REVERSIBLE covalent protein modification?
ex: phosphorylation
- undoable covalent bond
- kinase activity can add P back on while phosphatase removes P
117
What is the purpose of a MONOCYCLIC enzyme cascade?
- to covalently modify target enzyme (E)
118
What is the purpose of a BICYCLIC enzyme cascade?
- to covalently modify one of the modifying enzymes (F) that has an effect on target enzyme (E)
(may also have an effect directly on E)
119
How is glycogen metabolism under allosteric control?
- glycogen phosphorylase and glycogen synthase are under allosteric control
120
How is glycogen metabolism under covalent control?
Cascade phosphorylation (interconversion of enzyme forms)
121
What happens to glycogen phosphorylase in FASTING vs FED state?
FASTING: conversion from T to R by phosphorylation to break down glycogen
- activated by AMP
FED: conversion from R to T by dephosphorylation
- deactivated by ATP/G6P or glucose
122
What is the difference between allosteric and covalent control of glycogen phosphorylase?
COVALENT: adds P in T state when switching from fed to fasting
ALLOSTERIC: switches from T to R form to activate in fasting conditions
123
What does PHOSPHORYLASE KINASE a do?
- activates glycogen phosphorylase by phosphorylation
- inactivates glycogen synthase by phosphorylation
- 4 subunits
(a/b are covalently modified by PKA/PP1)
(gamma is catalytic)
(delta regulates Ca2+ sensitivity)
--> modulated by cAMP and Ca2+ release
124
What is PKA's effect on glycogen synthesis/breakdown?
- activates GPKa and PP1 inhibitor (stops activation of glycogen synthase), inactivates glycogen synthase
- bound by cAMP (ALLOSTERIC ACTIVATION) to activate, C unit dissociates and acts on target proteins
125
How is GLYCOGEN SYNTHASE modified?
Phosphorylated: less active
- PKA, phosphorylase kinase a, and glycogen synthase kinase
Non-phosphorylated: more active
- PP1c, low cAMP=low PKA
overall depends on % of enzyme in active vs non-active form
Allosteric: glycogen synthase B (inactive form) can be allosterically modified by G6P to promote dephosphorylation
126
What is PP1c?
- inhibits glycogen breakdown by dephosphorylating PKA, glycogen phosphorylase, and its own inhibitor PP1-inhibitor
-promotes glycogen synthesis by dephosphorylating G.S to activate it
127
How is PP1c controlled?
PP1-inhibitor, when phosphorylated, sequester PP1c which allow glycogen to be broken down
128
What regulated PP1-inhibitor?
1) PKA adds phosphate to turn it on
2) PP1c removes phosphate to turn it off
129
When FED, what is the state of PP1c in muscle?
- protein kinase is insulin stimulated which phosphorylates P1
- increased PP1c activity
- PP1c remains BOUND TO Gm subunit (on glycogen) leading to increased glycogen synthesis
130
When FASTING, what is the state of PP1c in muscle?
- protein kinase A stimulated by epinephrine which phosphorylates P2 (overrides P1)
- decreased PP1c activity
- PP1c detaches from Gm subunit, making PP1c inactive and leading to increased glycogen breakdown
131
What pathway governs insulin signalling?
- receptor tyrosine kinase pathway
132
What are the steps of the insulin signalling pathway regarding glycogen synthesis?
1) insulin binds receptor
2) kinase activation
3) glycogen synthase kinase is deactivated (phos.)
4) glycogen synthase remain on (dephos.)
133
What pathway governs glucagon/epinephrine signalling?
Gas receptor pathway
- B adrenergic receptors
134
What are the steps for the glucagon/epinephrine signalling pathway?
1) hormone binding causes GDP to be subbed with GTP and Ga dissociated from Gbg
2) activates adenyl cyclase
3) adenylate cyclase produces cAMP from ATP
4) PKA is activated by cAMP
5) PKA triggers cell response: turns ON glycogen phosphorylase, causing glycogen break down
135
What kind of signalling pathway is the Gaq receptor pathway?
A-adrenergic
136
Where does the Gas receptor pathway occur?
- muscle or liver
137
Where do insulin receptor pathways occur?
- muscle or liver
138
Where do glucagon receptor pathways occur?
- liver
139
Where do a-adrenergic receptor pathways occur?
- liver
140
What are the steps of the a-adrenergic pathway?
1) ligand binds and conform. change
2) PLC activated and hydrolyzes PIP2 and IP3 and DAG
3) IP3 stimulates Ca2+ release in the ER by binding its receptor
4) Ca2+ activates various cell processes through calmodulin
141
Why are a-adrenergic receptors not found in muscle cells?
- they already have a way of generating intracellular calcium currents (contraction), but liver cells don't so therefore they need the a-adrenergic receptors to do this
142
What do a-adrenergic receptors do and bind?
Bind: epinephrine
Outcome: prevent glycogen synthesis due to stress conditions
143
What is the outcome of GLUCAGON binding its receptors?
- only receptors on the liver
- causes increased glycogen degradation so that glucose can be released to blood
144
What is Hers' Disease?
Liver phosphorylase deficiency
- cannot properly catalyze breakdown to G1P from glycogen
- inability to breakdown glycogen in the liver
- causes HYPOGLYCEMIA
145
What is Von Gierke's Disease?
G6Pase deficiency
- cannot properly convert G6P to glucose
- cannot increase blood glucose in response to glucagon or epinephrine
- causes hypoglycemia and liver enlargement
146
What is McArdle's Disease?
Muscle phosphorylase deficiency
- glycogen breakdown is impaired in muscle so decreased fuel for glycolysis in muscle
- causes painful cramps during excercise
- leads to increased AMP because need G1P to gain phosphates to create ATP but G1P is produced by glycogen phosphorylase
147
What are the main steps of muscle contraction?
1) NT release activates ACh receptors on sarcolemma
2) AP generated down to T tubules
3) Triggers Ca2+ release from SR
4) Ca2+ binds to troponin, removing block of tropomyosin
5) allows contraction by myosin cross bridges
6) removal of Ca2+ by active transport into SR after AP
7) tropomyosin blockage returns and contraction ends
148
What leads to GLYCOGEN BREAKDOWN IN MUSCLE?
- epinephrine activates B-adrenergic receptors
- glycogen degradation leading to glycolysis
- activated phosphorylase kinase a to activate glycogen phosphorylase
- inhibition of glycogen synthase
149
What leads to GLYCOGEN BREAKDOWN IN LIVER?
- stress and epinephrine and glucagon
- phosphorylase kinase is activated which activates glycogen phosphorylase
- inhibition of glycogen synthase
- glucose is released to blood
150
What leads to GLYCOGEN SYNTHASE IN MUSCLE?
- insulin stimulates insulin receptors and GLUT 4 receptors
- glucose uptake is increased and glycogen synthesis is increased
- PP1c activated which activated glycogen synthase through dephosphorylation
- G6P activates glycogen synthase and inactivates glycogen phosphorylase
151
What leads to GLYCOGEN SYNTHASE IN LIVER?
- insulin stimulates insulin receptors to increase glycogen synthesis within the cell (NO GLUT4)
- increased PP1c activity increases glycogen synthase activity
- glucose and G6P inhibit glycogen phosphorylase
- G6P activates glycogen synthase
152
What is the purpose of the pyruvate dehydrogenase complex?
To convert pyruvate into acetyl coA
153
Where does the PDC reaction occur?
mitochondria
154
How does pyruvate get into mitochondria?
pyruvate translocase (H+ symport)
155
Why is H+ imported with pyruvate?
Because pyruvate has a (-1) charge so this keeps charge neutrality
156
What are the 3 subunits of PDC?
E1, E2, E3
157
What subunit of the PDC are PDK and PDP located on?
E1! Because this enzyme is covalently regulated
158
What is the irreversible step of the PDC?
the first step: decarboxylation of pyruvate, releases CO2 into the cytoplasm, preventing reversibility
159
What does E1 of the PDC do?
- decarboxylation of pyruvate
- irreversible
- attaches the remaining carbon molcule (C2&C3) to TPP
160
What does E2 of the PDC do?
- catalyzes steps 2 and 3
- reduces lipoamide and adds CoA to acetyl group to produce acetyl coA (step 3) and regenerate TPP (step 2)
161
What does E3 of the PDC do?
- catalyzes steps 4 and 5
- resets lipoamide by reducing FAD
- then reset FAD by generating NADH + H+ to be used in ETC
162
What are 3 advantages of a multi-enzyme complex?
1) minimized distances for substrates in between active sites (efficient)
2) intermediates are channeled between enzyme sites instead of side reactions occurring
3) coordinated control (shutting off one shuts off the whole thing)
163
What two enzymes control the utilization of pyruvate?
PDC (ox phos and CAC)
LDH (anaerobic lactate production)
164
What happens to PDC in cancer cells?
- suppressed by phosphorylation through PDK so that pyruvate is converted to lactate via LDH
165
How is the PDC controlled?
PRODUCT INHIBITION: by acetyl CoA and NADH (allosteric)
- pushes reaction in reverse
- prevents useless consumption of pyruvate
- reaction 1 never flips direction, but the rest do
COVALENT MODIFICATION: by E1 phosphorylation (turned off)
- PDP activates PDC
- PDK inactivates PDC (allosterically activated by NADH and Acetyl CoA
166
What is the effect of Ca2+ on the PDC?
- when exercising, Ca2+ is high, this activates PDP to increase PDC
- when resting, Ca2+ is low, activating PDK to decrease PDC
167
What is generated from the Citric Acid Cycle?
NADH, FADH2 and CO2
168
What is ANABOLISM?
- using CAC intermediates for anabolic pathways (ex: building blocks for FAs, etc...)
- CATAPLEUROTIC reaction: emptying CAC/depleting
169
What is CATABOLISM?
- forming CAC intermediates by breaking down more complex molecules (ex: breakdown AAs)
- ANAPLEUROTIC reaction: filling up CAC
170
What is the net production of the CAC?
3 NADH
FADH2
GTP or ATP
CoA
2CO2
171
How many ATP get produced per turn of the CAC?
1 NADH = 2.5 ATP (3) = 7.5
1 FADH = 1.5 ATP = 1.5
1 ATP = 1
total = 10 ATP
172
What are the 3 irreversible steps of the CAC?
1) citrate synthase
3) isocitrate dehydrogenase
4) a-ketoglutarate dehydrogenase
173
What kind of regulation are the RLS of the CAC under?
allosteric (not covalent)
174
How many ATP are produced in total from respiration?
GLYCOLYSIS : 7 ATP
PDC: 2.5 ATP (x2)
CAC: 10 ATP (x2)
total = 32 ATP
175
Which are the "high-energy" steps of the CAC?
steps 1-5
176
What limits the forward reaction of STEP 1 of the CAC?
energetically : Acetyl CoA (b/c of thioester bond = high E)
abundantly: oxaloacetate
177
What is special about a-ketoglutarate?
it is a 5C molecule (releases CO2) and this is an entry point for anapleurotic pathways (refill)
178
What is step 4 of the CAC similar to?
PDC! (just ketoglutarate dehydrogenase instead)
- same subunits
- generates succinyl-coA
179
Why is step 5 able to produce an ATP/GTP?
succinyl-coA forms a high energy bond so breaking that releases a lot of E that can be used and coupled to the formation of GTP or ATP
180
What is STEP 6 of the CAC?
COMPLEX II of the ETC
- covalently bound to FAD
- restores FAD by funnelling electrons into the ETC
- no protons pumped by complex II, just donates electrons to Q pool
- produces fumarate
181
Although step 8 is highly unfavourable in vitro, why does it proceed in vivo?
it produces oxaloacetate which is of very small abundance compared to malate, so therefore the reaction will proceed forward
- in addition, the next reaction is very favourable b/c of acetyl CoA, so it will pull oxaloacetate forward in cycle
182
What activates or inhibits CITRATE SYNTHASE (1)?
1) substrate abundance
2) product inhibition
3) competitive feedback by succinyl-coA
4) allosteric inhibition (NADH)
5) allosteric activation (ADP)
183
What activates or inhibits ISOCITRATE DEHYDROGENASE (3)?
1) Product inhibition (NADH)
2) Allosteric Activation (ADP & Ca2+)
184
What happens when isocitrate dehydrogenase is off?
Isocitrate goes back to citrate which can leave the mitochondria to the cytoplasm and activate acetyl coA carboxylase, FA synthesis (catapleurotic), inhibits glycolysis & PFK
185
What activates or inhibits a-KETOGLUTARATE DEHYDROGENASE?
1) Product inhibition: NADH, succinyl-CoA
2) Allosteric activation: Ca2+
186
Why does the PDC get switched off?
When energy charge is high, product inhibition by Acetyl-CoA and NADH, also activates PDK
187
What is PYRUVATE CARBOXYLASE?
- converts pyruvate into oxaloacetate
- allosterically activated by Acetyl-CoA (builds up when no oxaloacetate to combine with)
- allows for a restart of the CAC
188
What causes CITRATE EFFLUX to cytosol?
- high energy charge
- stimulates the biosynthesis of FAs and inhibits PFK (downregulates glycolysis)
189
What is a CATAPLEUROTIC REACTION?
- syphoning off of the CAC intermediates
- anabolism using CAC intermediates
190
What is an ANAPLEUROTIC REACTION?
- replenishing of depleted CAC intermediates
- catabolism using CAC intermediates
191
What type of reaction does PEPCK catalyze?
CATAPLEUROTIC
- removes carbon from oxaloacetate and gives it to PEP to form glucose
192
What does OXALOACETATE TRANSAMINATION do?
CATAPLEUROTIC
- converts oxaloacetate to aspartate
- adds amine group to oxaloacetate
- this can form other AAs, etc that can exit to cytosol
193
What is formed by CITRATE BREAKDOWN?
CATAPLEUROTIC
- FAs, steroids
- these can be moved out to cytosol
- they can be reused to form Acetyl-CoA and oxaloacetate in cytosol
194
What are the main 4 CATAPLEUROTIC reactions of the CAC?
1) PEPCK
2) OXALOACETATE TRANSAMINATION
3) CITRATE BREAKDOWN
4) a-KETOGLUTARATE TRANSAMINATION
195
What does a-KETOGLUTARATE TRANSAMINATION do?
- converts a-KG to glutamate by adding an amine group
- this can be converted to other AAs or purines
196
What is the central hub for amination?
GLUTAMATE
- donates amines to other transamination reactions
- a-KG picks up amines from other transamination reactions to form glutamate
197
What are the main ANAPLEUROTIC REACTIONS in the CAC?
1) Pyruvate carboxylase
2) Malic enzymes
3) Transamination pairs
4) Glutamate dehydrogenase
198
What does PYRUVATE CARBOXYLASE do?
- replenishes oxaloacetate by adding a C to pyruvate
199
What do MALIC ENZYMES do?
- make malate from pyruvate (but opposite is more important)
malate --> pyruvate produces NADPH for FA synthesis (catapleurotic)
200
What are the TRANSAMINATION PAIRS?
glutamate => a-ketoglutarate
aspartate => oxaloacetate
alanine => pyruvate
201
What does GLUTAMATE DEHYDROGENASE do?
removes an amine from glutamate and releases it as ammonium (urea cycle) NOT TRANSAMINATION
202
What occurs in the mitochondrial inner membrane and the matrix and the outer membrane?
INNER MEMBRANE: ETC, ox. phos.
MATRIX: CAC, PDC, FA ox., urea cycle, glutamate dehydrog.
OUTER MEMBRANE: phospholipid synthesis
203
What drives the transport of electrons in the ETC?
- the electrons move from LOW to HIGH reduction potential from complex 1 -->4 (low = 1 and high = 4)
204
What are some examples of redox centres in the ETC?
- flavins (ex: FAD)
- Fe-S clusters
- cytochromes with heme
- copper
205
What happens in complex 2 of the ETC?
- no protons pumped
- enzyme in CAC
- takes electrons from succinate and uses it to reduce Q through heme
205
What happens in complex 1 of the ETC?
- net 4H+ pumped
- H+ from NADH (CAC) reduce Ubiquinone
- Nqo12 subunit is coupled to Q reduction, so when that happens, 4 H+ get pumped across the membrane
206
How can Q get reduced from the matrix?
fatty acyl CoA --> FAD --> ETF -->Q
207
How can Q get reduced from the inter-membrane space?
glycerol-3-P --> FAD --> CoQ
208
What happens in complex 3 of the ETC?
- net 4H+ pumped
- Q cycle occurs here (reduction of Cyt C)
- NET: 2 Cytc-H, 2Q (ubiquinone) and 1QH2 (ubiquinol)
209
What happens in part 1 of complex 3 (Q cycle)?
- 1st electron reduces Cyt C
- second electron goes to Q to form QH (semi-quinone)
210
What happens in part 2 of complex 3 (Q cycle)?
- 1st electron reduces Cyt C
- second electron goes to QH to form QH2 (ubiquinol)
211
What happens in complex 4 of the ETC?
- net 2H+ pumped
- copper reduction centres here (highest reduction potential)
212
What makes up the RESPIRASOME?
- two subunit I
- two subunit II
- two subunit IV
- one subunit III in the middle
213
Why are so many redox reaction required in the ETC?
- most of the E is harnessed and converted to a stored form so only a little bit can be used
214
What is the P/O ratio?
the number of ATP molecules that are produced per molecule of O2 consumed during oxidative phosphorylation
215
What is the P/O ratio through COMPLEX 1?
10/3.7 = 2.5
216
How do we calculate the P/O ratio?
In humans, we produce 3 ATP per 360* turn and it uses 8H+ because there are 8 ST subunits.
This gives 2.7 H+ per ATP. We need to include on more H+, because to bring in a Pi for ATP we must bring in another H+ as well. So, since we use 10H+ to pump across the membrane and we need 3.7 per ATP we have 10/3.7 = 2.5
217
How does the P/O change if we go through complex 2?
We pump 4 less protons so that ratio will be 6/3.7 instead, giving a P/O ratio of 1.5
218
What is the F0/F1 ATPase?
When ATP synthase goes in reverse! (not likely in vivo)
219
Are RESPIRATION and OX. PHOS. the same?
NO! You can have respiration without ox. phos. Respiration is glycolysis and TCA, while ox. phos. is ETC and ATP synthase
220
What is the DHAP/G3P shuttle?
- brings electrons from cytosol into the ETC
- DHAP is reduced by NADH in the cytosol
- G3P is then shuttled into the inner membrane of the mitochondria
- G3P donates its electrons to reduce FAD --> FADH2 (in complex 2) and reforms DHAP
- DHAP is then shuttled back out of the membrane to cytosol
221
What is the MALATE/ASPARTATE shuttle?
-oxaloacetate is reduced to malate in the cytosol
- malate is carried into mitochondria inner membrane
- malate is oxidized to oxaloacetate (reducing NADH)
- oxaloacetate undergoes transamination to aspartate using glutamate's amine
- aspartate and a-KG are then transported back to the outer membrane
- these two are then reconverted to oxaloacetate and glutamate
222
What is the P/O for complex 1?
4/3.7 = 1
223
What is the P/O for complex 3?
2/4 = 0.5 (because although it pumps 4, two are given to CoQ)
224
What is the P/O for complex 4?
4/3.7 = 1 (because although it pumps 2, two are also lost to H2O)
225
What is the correct theory of the respiration vs the ETC?
CHEMI-OSMOTIC THEORY: ETC and phosphorylation are coupled due to this type of mechanism
226
What does DNP do?
Uncouples the ETC from ATP synthase
- releases H+ from the IMS back into the matrix to deplete the proton gradient and increase respiration (w/o ox. phos.)
- maybe a weight-loss mechanism
227
How many proteins come from mtDNA?
13!
228
Compare mitochondrial DNA-encoded proteins and nuclear DNA-encoded proteins in the ETC.
COMPLEX 1: 7 mito/36 nuclear
COMPLEX 2: 0 mito/4 nuclear
COMPLEX 3: 1 mito (cytb)/ 10 nuc
COMPLEX 4: 2 mito/14 nuclear
229
How is cellular respiration controlled?
When mitochondria are functioning properly you won't consumer O2 if you don't need to --> NO WASTE
If ADP concentration increases, more ADP will be phosphorylated to ATP which moves H+ into the matrix (relieves thermodynamic back pressure), causing an increase in respiration
230
What happens when ADP +Pi is injected into the cell along with glutamate?
Respiration increases due to increased O2 consumption rate x3!
- Glutamate is needed as it replenishes CAC intermediates (anapleurotic)
231
What happens to respiration if you double the injected ADP concentration?
- double the oxygen is consumed, but it is consumed at the same rate
232
How is dG' calculated?
dG' = -n(F)(dE')
where n = the number of electrons
F = 96.4 kJ/V/mol
dE' = (E'e accept - E'e donor)
232
What does OLIGOMYCIN do?
Inhibits ATP synthase which decreases respiration because H+ gradient isn't being depleted
233
What is the F1 component of complex V?
- located in the mitochondrial matrix
- 3 ADP binding sites (Bdp) and 3 ATP production sites (Adp)
234
How to calculate H+ used per ATP?
- divide the number of protons translocated (c subunits = 8) by the number of ATP produced (3) = 8/3 = 2.7 H+ per ATP
235
How does the Pi get into the IMS from the matrix for ATP production?
PHOSPHATE CARRIER: bring a Pi in as well as an H+ (relies on ETC) to maintain neutrality
236
How does the ADP get into the IMS for ATP production?
ADP/ATP carrier
- brings ATP4- into IMS
- releases ADP3- into matrix
- matrix is more (-) than IMS, so this draws the ATP(-) into the IMS
237
What is the main goal of the ADP/ATP carrier?
- releases ATP into the IMS
238
What drives the ADP/ATP carrier?
the charge gradient (more + inside IMS) draws the ATP4- into the IMS
239
What are the c and m states of the ADP/ATP carrier?
C state => facing IMS
- ADP can bind
- transition state
- ADP released to matrix
M state => facing matrix
- ATP can bind
- transition state
- ATP released to the IMS
240
What inhibits the ADENINE NUCLEOTIDE TRANSLOCASE?
- inhibited by atractyloside and CATR (fixes it in place)
241
What are the three parts of the ADENINE NUCLEOTIDE TRANSLOCASE?
1) phosphate carrier
2) ADP/ATP carrier
3) ATP synthasef
BIOC 311
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