8) Metabolism Flashcards
(90 cards)
1
Q
Define metabolic pathways
A
A series of enzymatic reactions producing specific products - branched and interconnected
2
Q
What are metabolites
A
Reactants, intermediates and products
Varies by cell type, nutritional status and developmental stage
3
Q
What is metabolic flux
A
Living systems maintain a steady state of flux through a metabolic pathway. Rates of synthesis and breakdown of metabolites maintain concentrations - a steady state far from equilibrium allows flux
Flux is determined by the rate determining Step of pathway
Cells control flux through these rate-determining steps by both short and long term strategies
4
Q
Describe degradative pathways
A
Breakdown of product to create energy
- often converge on common intermediates eg 2 C - unit of acetyl CoA
- further metabolised in central oxidative pathway eg citric acid cycle
5
Q
Describe biosynthetic pathways
A
Create metabolites
- carry out the opposite of degradative pathways
- few metabolites are the starting points
6
Q
How is metabolic flux controlled
A
Allosteric control:
- enzymes can be regulated by effectors (substrates, products, coenzymes)
Covalent modification:
- enzymes may have specific regulatory sites
- may be subject to hormonal control
Substrate cycles:
- vary the rates of 2 opposing non-equilibrium reactions
Genetic control:
- protein synthesis rates can affect enzyme amounts and activities
- changes within hours or days so a long term control mechanism
7
Q
Where do we source energy from in our diet
A
Energy sources are carbohydrate, fat (lipid) and protein (2000-3000 kcal / day)
- carbohydrate and protein yield about 4kcal (17kJ) of energy per gram, lipid yields 9
- recommended intake is 30% of total calories as fat, 15% protein, 55% carbohydrate
8
Q
Summary of glucose as a molecule
A
Most abundant carbohydrate in human body
- exists in D and L enantiomers
- found in plasma and cells
- stored in human tissue as insoluble polymer - glycogen (starch in animals)
- comes from diet or body stores
- in solution forms a 6 member Ed ring structure, a pyranose (5 membered rings are furanose)
9
Q
How is glucose utilised
A
Major transported in carbohydrates in blood
- blood conc tightly controlled by hormones (insulin and glucagon)
- fasting concentration -4mM rises to -8mM following high carb meal
- required by brain and erythrocytes
10
Q
Overview of glycolysis
A
- employed by all tissues for glucose oxidation to provide energy (ATP)
- ‘all’ sugars can be ultimately converted to glucose with an adequate supply of oxygen (+ mitochondria)
- pyruvate is the end product
- ‘aerobic’ glycolysis as O2 required to re-oxidise NADH
- oxidative decarboxylation of pyruvate to acetyl-CoA > TCA
- ‘anaerobic’ glycolysis (no oxygen or mitochondria)
- pyruvate is reduced to lactate as NADH reoxidised
11
Q
How does glucose enter the cell
A
2 methods:
1) Na+ independent facilitated diffusion transport
2) ATP dependent NA+ - monosaccharide transport
12
Q
How does glucose enter the cell by Na+ independent facilitated diffusion transport
A
- glucose moves via concentration gradient
- GLUT 1 to 14
- these transporters exhibit tissue-specific expression
- GLUT 4 is common in muscle and adipose
13
Q
How does glucose enter the cell by ATP - dependent Na+ - monosaccharide transport system
A
- a ‘’co transport’’ system transports glucose against a gradient (coupled to Na+ gradient)
- found in intestinal epithelial cells
14
Q
What are the 2 stages of conversion of glucose to pyruvate
A
1) ‘energy investment phase’ ( first 5 reactions)
- phosphorylated forms created using ATP
2) ‘ energy generation phase’
15
Q
Describe the first glycolytic reaction in most tissues
A
- glucose <> glucose 6-P
- glucose phosphorylation is catalysed by hexokinase (I-III) in most tissues (uses ATP)
- 1 of 3 regulatory enzymes of glycolysis inhibited by glucose 6 phosphate
- low Km (high affinity for glucose)
- permits efficient phosphorylation in low [glucose]
- low Vmax means no over abundance of glucose 6-phosphate
16
Q
Describe the first glycolytic reaction in the liver
A
Glucokinase (hexokinase IV): liver parenchymal cells / B cells ]
- higher Km so only active following consumption of a carb rich meal
- high Vmax allowing glucose delivered to liver to be maximally absorbed
17
Q
Describe 2nd glycolytic reaction (isomerisation)
A
Isomerisation of glucose 6-phosphate to fructose 6-phosphate
- catalysed by phosphoglucose isomerise
- readily reversible and not rate limiting
18
Q
Describe the 3rd glycolytic reaction (another phosphorylation)
A
Fructose 6-phosphate phosphorylation
- an irreversible reaction, rate limiting, catalysed by phosphofructokinase -1
- the most important control point
19
Q
Which enzymes catalysed the phosphorylation of fructose -6- phosphate and how does it work
A
Phosphofructokinase-1 (PFK-1)
- controlled by [ATP] and [fructose 6-phosphate]
- high [ATP]= inhibition and high [AMP] = activation : shows abundance of energy available
- also inhibited by citrate, TCA intermediate
- favours glycogen synthesis
20
Q
Describe fructose 1,6-biphosphate cleavage (no 4 and 5)
A
- aldolase cleaves fructose 1,6-biphosphate to DHAP and glyceraldehyde 3-phosphate
- reversible and unregulated reaction
- triode phosphate isomerise allows inter conversion
- only glyceraldehyde 3- phosphate can be used by glycolysis
- DHAP is used in triacylglycerol synthesis
21
Q
Describe 6th and 7th reaction of glycolysis
A
The first oxidation- reduction reaction of glycolysis follows:
- phosphate group os also attached (drives the synthesis of ATP in next reaction)
No 7 synthesis of 3-phosphoglycerate, produces ATP
- 2 molecules of 1,3-BPG are formed for each glucose
- 2 ATP produced replaces ATP consumed earlier
Catalysed by the physiologically reversible enzyme
22
Q
Describe glycolysis reactions 8-10
A
Phosphoglycerate mutase shifts the phosphate from carbon 3 to 2
- a reversible reaction
Enolase then redistributes the energy within the molecule by dehydration (a reversible reaction)
- another high energy intermediate
Pyruvate formation linked to ATP production catalysed by pyruvate kinase
- 3rd irreversible reaction of glycolysis
- an example of substrate - level phosphorylation
23
Q
How is anaerobic glycolysis different to aerobic
A
In aerobic conditions (strenuous exercise) the respiratory chain cannot oxidise NADH to regenerate NAD+ (requires O2)
- glycolysis will convert NAD+ to NADH but NAD+ is required for glycolysis to continue
- pyruvate reduced to lactate by lactate dehydrogenase (LDH) to regenerate NAD+
- different LDH isozymes in different tissues
24
Q
Overall energy yield from glycolysis
A
ATP yield anaerobic conditions = +2ATP
Aerobic conditions yield additional 6 ATP from oxidation of 2NADH
25
how is haemolytic anemia caused
Mature red blood cells lack mitochondria
- dependent on glycolysis for ATP generation
- fuel ion pumps to maintain shape
- failure to generate ATP: cell shape changes and phagocytosis
Genetic defects of glycolytic enzymes lead to haemolytic anemia
26
Describe glycogen, its storage and what its used for
Mobilisation from glycogen (glycogenolysis)
Main stores are found in skeletal muscle and liver
- 400g (1-2% fresh weight) in muscle
-100g (10% fresh weight) in liver
Some alterations in glycogen storage diseases
Mobilisation of glucose from glycogen stores in liver (and kidney)
In muscle it serves as the fuel reserve for ATP synthesis
27
How many reducing and non reducing ends does glycogen have
Has one reducing end but a non reducing end on every branch (so rapid mobilisation)
28
Name the 3 cytosolic enzymes required for glycogen degradation
1) glycogen phosphorylase
2) glycogen debranching enzyme
3) phosphoglucomutase
29
Describe the structure and action of glycogen phosphorylase
- Dimer (97kD)
- cleanses a (1-4) bonds until 4 glycosyl units remain on a branch point
- a limit dextrin
Enzyme activity:
- allosteric interactions and covalent modification:
- inhibitors : ATP, G6P, glucose / activator: AMP
Induces conformational change (reveals buried active site)
30
Describe the structure and function of glycogen debranching enzymes
Enzyme is bifunctional with 2 active sites
- acts as a(1-4) transglycosylase
Moves trisaccharide units to non-reducing branch end
- then hydrolytically removes the remaining glycosyl using amylo-a(1-6)-glucosidase activity
31
Describe the structure and function of phosphoglucomutase
Glycogen phosphorylase converts the glycosyl units of glycogen to G1P
We then see a phosphorylation of the glucose molecule followed by a re-phosphorylation of the enzymes
- G6P can then continue along the glycolytic pathway or the pentode phosphate pathway it can be hydrolysed by glucose -6- phosphatase to glucose
32
How is glycogen synthesised
G6P produced by gluconeogenesis may not be hydrolysed to glucose but may be converted to G1P for incorporation into glycogen
- glycogen synthase makes a(1-4) linkages only extends existing chain, so glycogenin attaches to glucose
- this would be a linear compound like amylose but that used ADP-glucose)
Glycogen synthesis accelerates after a meal (the well fed state)
33
How does the rate of glycogenolysis and glycogenesis change in the liver
Glycogenesis accelerates during well fed periods
| Glycogenolysis accelerates during fasting
34
On which 2 levels does regulation of glycogenesis and glycogenolysis occur
Glycogen synthase and glycogen phosphorylase are horomonally regulated via phosphorylation / dephosphorylation
- glycogen synthase and glycogen phosphorylase are allosterically regulated
35
How is glycogen metabolism regulated by hormones
Insulin, glucagon and adrenaline:
- hormones act through changes to phosphorylation state of enzymes
- binding of hormones to cell surface receptor triggers intracellular events
- adrenaline and glucagon act through a ‘second messenger’ - cyclic AMP
- adrenaline acts on muscle and liver
- glucagon acts on liver
36
How is glycogen metabolism regulated allosterically
As well as hormones, glycogen synthase and glycogen phosphorylase respond to metabolites and energy needs
- gluconeogenesis is inhibited when substrate and energy levels are high
- glycogenolysis is increased when glucose and energy levels are low
- permits rapid response
- can override hormone - mediated covalent regulation
37
How is glycogen metabolism regulated allosterically in the high energy state
[glucose] high, [G6P] high, [ATP] high
Glycogen phosphorylase inhibited by G6P and ATP (and glucose in liver)
Glycogen synthase activated by G6P
Glycogen synthesis predominates
38
How is glycogen metabolism regulated allosterically in the low energy state
[glucose] low, [G6P] low, [ATP] low, muscle [AMP] high
Muscle glycogen phosphorylase activated by AMP (raised by contraction)
Glycogenolysis predominates
39
How is calcium involves in glycogenolysis
- glycogenolysis in muscle is activated by calcium
Released into cytoplasm after neural stimulation released from sarcoplasmic reticulum
- muscle contraction results from increased cytosolic calcium
Calcium binds to and activated calmodulin (Ca2+ binding protein)
Subunit of phosphorylase kinase
- activates complex b
- phosphorylase kinase phosphorylates and activates glycogen phosphorylase
40
What is glycogen storage disease
Genetic diseases caused by defects in enzymes required for degradation or synthesis
- glycogen has abnormal structure or in accumulation of excessive amounts
- can range from fatal in early childhood to mild disorder
41
Where does TCA cycle occur
Mitochondrial matrix
42
What is the TCA cycle involved in
Energy production- removal of pairs of electrons to form NADH+, H+ and FADH2 from NAD+ and FAD+
- biosynthesis of metabolites
43
What is the importance of the PDH reaction
Commits pyruvate to TCA cycle
- controls the entry of glucose to TCA cycle
- rate limiting step
- irreversible
- regulated
- allosterically
- covalently
- hormonally (insulin activates)
44
Give overview of structure of PDH
Multi enzyme complex
Molecular mass 4-10 million daltons
Consists of 3 enzyme complexes
5 coenzymes
45
3 enzyme activities of PDH
E1- pyruvate decarboxylase
E2- dihydrolipoyl transacetylase
E3- dihydrolipoyl dehydrogenase
46
5 coenzymes in the PDH complex
-Thiamine pyrophosphate (TPP)
thiamine (vit B)
- lipoamide
Lipoic acid - 10 C fatty acid with sulphydryl groups on C8 and C10
- CoA
- pantothenic acid (vit B5)
FAD+
Riboflavin (vit B2) prosthetic group
- NAD+
Niacin (vit B3)
47
Summarise mechanism of PDH
1) pyruvate is decarboxylated to form a hydroxyethyl derivative bound to the reaction carbon of thiamine pyrophosphate, the coenzyme of E1
2) the hydroxyethyl intermediate is oxidised by transfer to the disqualified form of lipoic acid covalently bonded to E2
3) the acetyl group, bound as a thioester to the side chain of lipoic acid is transferred to CoA
4) the sulfhydryl form of lipoic acid is oxidised by FAD dependent E3, regenerating the disulfide form of lipoic acid
5) FADH2 on E3 is reoxidised to FAD as NAD+ is reduced to NADH and H+
48
What medical problems are associated with PDH
- beri beri (thiamine deficiency) damage to PNS and weakened muscle common in Far East
- mercury and arsenic poisoning binds to dihydrolipoyl groups on E2, CNS pathologies
- vitamin deficiencies riboflavin and niacin are components of FAD and NAD
- pyruvate dehydrogenase deficiency, lacticacidemia (anaerobic carbohydrate metabolism), reduced ATP synthesis (NB in CNS) elevated alanine
49
In the TCA cycle, describe the citrate synthesis reaction
- oxaloacetate + acetyl CoA > citrate
- enzyme is citrate synthase, inhibited by citrate
- irreversible condensation reaction
- uses 1 H2O and releases 1 CoA
50
In the TCA cycle describe the citrate isomerisation reaction
Citrate is isomerised to isocitrate through hydroxyl group migration
Catalysed by aconitase
51
In the TCA cycle describe the oxidative decarboxylation of isocitrate
Isocitrate is converted to alpha-ketoglutarate in an irreversible oxidative decarboxylation reaction
- enzyme is isocitrate dehydrogenase
- yields on NADH molecule and one CO2
- rate limiting step: enzyme is allosterically activated by ADP (low energy signal) and Ca2+ and is inhibited by ATP and NADH (high energy signal)
52
In the TCA cycle describe the oxidative carboxylation of alpha-ketoglutarate
Alpha ketoglutarate is irreversibly converted to succinylcholine CoA
- catalysed by alpha-ketoglutarate dehydrogenase, a multi enzyme complex similar to PDHC
- it is inhibited by NADH and succinyl CoA, and is activated by Ca2+
- one NADH is produced and also one CO2
53
In the TCA cycle describe the succinyl coenzyme cleavage reaction
-succinyl CoA is cinverted to succinate
- enzyme is succinyl CoA synthase
- reaction uses Pi and GDP & GPT and CoA are produced
Reaction is reversible
54
In the TCA cycle describe the succinate oxidation reaction
Succinate is oxidised to fumarate
- coenzyme FAD is reduced to FADH2
- enzyme is succinate dehydrogenase, embedded in the inner mitochondrial membrane
- reaction is reversible
55
In the TCA cycle describe the reaction of fumarate hydration
Fumarate is hydrated to malate
- reversible reaction requiring H2O
- enzyme is fumarase
56
In the TCA cycle describe the reactions of malate oxidation
Malate is oxidised to form oxaloacetate (OAA)
- enzyme is malate dehydrogenase and reaction is reversible
- one NADH is produced
57
Describe the order of the TCA cycle starting with citrate synthesis
1) citrate synthesis
2) citrate isomerisation
3) oxidative decarboxylation of isocitrate
4) oxidative decarboxylation of alpha-ketoglutarate
5) succinyl CoA cleavage
6) succinate oxidation
7) fumarate hydration
8) malate oxidation
58
List the products of each stage of the TCA cycle starting with citrate
```
(Acetyl CoA)+ citrate
Isocitrate
Alpha-ketoglutarate
Succinyl CoA
Succinate
Fumarate
Malate
Oxaloacetate
```
59
What is the overall yield of the TCA cycle for each molecule of acetyl CoA oxidised
3 NADH + 1 FADH2 + 2CO2 + 1GTP ( 4 pairs of electrons)
60
At which stages is the TCA cycle regulated
Pyruvate to acetyl CoA
- isocitrate to alpha - ketoglutarate
- alpha - ketoglutarate to succinyl CoA
61
What are the 4 basic types of metabolic pathway
- fuel oxidative pathways
- fuel storage and mobilisation
- biosynthetic pathways
- detoxification / waste disposal pathways
62
What do anabolic pathways do
‘Anabolic’ pathways- synthesise large molecules eg biosynthetic and fuel storage pathways
63
What do catabolic pathways do
‘Catabolic’ pathways - breakdown large molecules eg fuel oxidation pathways
64
What is metabolic homeostasis
The control of the balance between substrate availability and need manifested by anabolic vs catabolic pathways
- a balance must be met between:
- intake of fats, protein, carbohydrates
- oxidation rates
- de novo synthesis
- rate of mobilisation from storage
65
What is the consequence of metabolism
Significant decreases (<60mg/dL) limit brain metabolism
- hypoglycaemia
- glucose influx lowers due to Km of blood brain barrier transporters
Too much: hyperosmolar effects = neurologic deficits and coma
- conc rises above renal tubular threshold
- non enzymatic glycosylation of proteins
66
What is the ideal range for blood glucose levels
80-100 mg/ dL (-5mM)
67
2 major metabolic hormones that regulate fuel storage and mobilisation
Insulin: promotes storage of fuels (& use for growth)
Glucagon: promotes mobilisation
68
Which tissues does insulin act on and what does it promote
Insulin acts on 3 main tissues: liver, muscle and adipose. Promotes:
- glycogen formation in liver and muscle
- conversion of glucose to triacylglycerols (liver)
- protein synthesis (eg albumin) in liver
- storage of triacylglycerols (adipose)
- increases glucose uptake by muscle and adipose
- AA uptake and protein synthesis in skeletal muscle
69
What does glucagon do, where does it act and what does it promote
Glucagon acts to maintain fuel availability
- principally acts in liver and adipose
- not muscle, due to lack of receptors
Promotes:
- increased glycogenolysis, reduced glycogen synthesis in liver
- stimulates gluconeogenesis, reduced glycogen synthesis in liver
- stimulates gluconeogenesis and ketogenesis
- mobilises fatty acids from adipose triacylglycerols
70
Where are insulin and glucagon produced
The pancreas is the site of production
- a cells secrete glucagon
- decrease plasma [glucose] and increase [adrenaline]
- B cells secrete insulin and increase plasma [glucose]
71
What is the structure of insulin
A polypeptide synthesises as a preprohormone (proinsulin)
- active form is around 51 amino acids
- central section (C peptide, white part) is cleaved and released to leave A and B chain connected by the disulfide bonds
72
How is insulin released
ATP generated by glucose metabolism closes K+ channels and depolarises B cell membrane
- opens voltage gated Ca++ channels
Ca++ influx stimulates insulin secretion
73
When is insulin released
- 80mg / dL threshold
- proportional release up to 300 mg/dL
- rapidly degraded by liver
- autonomic nervous system has a role
- vagus nerve stimulated in response to a large meal
74
Describe the structure of glucagon
- 29 amino acid chain
| - produced as a preprohormone in the RER
75
Where is glucagon degraded
Degraded by liver and kidneys (-5 min 1/2 life)
76
What is the secretion of glucagon regulated by
Secretion is regulated by [glucose] and [insulin]
| - levels build as both fall
77
Where is insulin degraded
Liver, muscle, kidney
78
What mechanisms does glucagon use to stimulate glucose production
- via glycogenolysis
| - via gluconeogenesis (from aa etc)
79
What happens inside the cells when insulin or glucose binds
Hormones change flux of substrates through pathway
- increase or decrease rates of slowest steps eg enzyme or transport protein rates
- these hormones bind receptors on a cells surface
- initiates intracellular signalling cascades
- second messengers activated
- signal ‘transduction’
80
3 types of signal transduction
1) receptor coupled to adenylate cyclise produces cAMP
2) receptor / kinase activity
3) receptor coupled to hydrolysis of phosphatidylinositol biphosphate (PIP2)
81
What happens when insulin binds to a cell
Insulin autophosphorylates cell receptor
- receptors tyrosine kinase domain phosphorylates enzymes
- still trying to define full signalling events
Basic cellular responses:
1) reverses glucagon-stimulated phosphorylation
2) kicks off a phosphorylation cascade
3) induction / repression of enzyme
4) stimulate protein synthesis
5) stimulate glucose and aa intake
82
What happens when glucagon binds to a cell
Glucagon binding causes secondary messenger formation:
- G protein dissociation activates adenylate cyclase
- creates cAMP
- activates cAMP-dependent protein kinase
- phosphorylates regulatory enzymes
- controls carbohydrate and lipid metabolism
83
Where are glucagon receptors missing
Skeletal muscle
84
What are the important principles of signalling in glucagon intracellular events
- amplification of 1 degree signal
- integration of metabolic processes eg glucagon
- phosphorylation of enzymes
- activates glycogen degradation
- inhibits glycogen synthesis
- inhibits glycolysis
- rapid termination of signal
85
When is the well fed state
The 2-4 hours period after eating a meal
86
What happens to the fuels we ingest as food
These fuels are oxidised for the body’s energy needs
- energy is transported to storage sites
- readily available substrates
- anabolic state
- increased triacylglycerol and glycogen synthesis
87
What is oxidation and storage of fuels determined by
The insulin / glucagon ratio
88
How are dietary carbohydrates processed by the body after ingestion
Dietary carbohydrates are turned into monosaccharides
- starch is digested by a-amylase
- disaccharides / trisaccharides / oligosaccharides digested by enzymes in the brush border (intestine)
- monosaccharides absorbed by intestinal epithelial cells
- transported to the hepatic portal vein
- glucose is oxidised for energy and enters biosynthetic pathways
- forms the carbon skeleton of most compounds eg triacylglycerols
89
How are proteins processed by the body after ingestion
Proteins are cleaved by pepsin in the stomach and proteolytic enzymes in the pancreas.
- absorbed into intestinal epithelial cells
- released into hepatic portal vein
- free amino acids absorbed from blood used for protein synthesis and biosynthesis eg neurotransmitters and heme
- carbon skeleton may be oxidised
90
How are fats processed by the body after being ingested
Fats aren’t soluble so digestion is problematic
- triacylglycerols are the major lipids of the diet
- emulsified by bile salts
- pancreatic lipase converts TAGs to fatty acids and 2-monoacylglycerols
- forms micelles contacting with bile salts
Absorbed and then reformed into TAGs
- TAGs packaged with proteins, phospholipids, cholesterol into chylomicrons
- secreted into lymphatic system
- enter bloodstream via thoracic duct
