Additional Pathways of Carb Metabolism

Question 1

What is gluconeogenesis?

Answer 1

The synthesis of glucose from non-carbohydrate precursors
Almost the reverse of glycolysis as it can by the pathway from pyruvate to glucose

Mainly occurs in the liver (only a little in the kidney)

Question 2

What are the non-carbohydrate precursors that can be converted back into glucose?

Answer 2

Lactate, pyruvate, citric acid cycle intermediates and the carbon skeletons of most amino acids

Question 3

What are the non-carbohydrate precursors initially converted into?

Answer 3

A common intermediate - oxaloacetate
(however, leucine and lysine cannot be converted to oxaloacetate, as their breakdown will produce acetyl-CoA)
Oxaloacetate is also a citric acid cycle intermediate
COO CH2 CO COO

Question 4

Which stages of gluconeogensis are different in reverse to glycolysis?

Answer 4

Pyruvate -> phosphoenolpyruvate

Fructose-1,6-bisphosphate -> fructose-6-phosphate

Glucose-6-phosphate -> Glucose

Question 5

What is step 1 of gluconeogensis?

Answer 5

Pyruvate is converted into phosphoenolpyruvate in two steps
Pyruvate -> oxaloacetate
Uses pyruvate carboxylase and ATP
Oxaloacetate -> phosphoenolpyruvate (PEP)
uses PEPCK and GTP
This is exothermic decarboxylation as CO2 is released

Question 6

Step 1 gluconeogenesis - describe pyruvate carboxylase?

Answer 6

Tetrameric protein
Uses a biotin cofactor to facilitate catalysis
Biotin functions as a CO2 carrier
Biotin is a cofactor derived from vitamin B7 or H

Question 7

Step 1 gluconeogenesis - what does pyruvate carboxylase do?

Answer 7

It has a two phase reaction:
Cleavage of ATP drives dehydration of bicarbonate to form high energy carboxyphosphate
Resulting CO2 reacts with biotin to yield an activated intermediate
Activated carboxyl group is transferred to pyruvate to form oxaloacetate

Two reaction phases occur on different sub-sites of the enzyme
The CO2 carrying biotin ring is transferred between the sites on its flexible arm

Question 8

Give an overview of the enzyme positions in step 1 of gluconeogenesis?

Answer 8

Gluconeogenesis requires metabolite transfer between mitochondria and cytosol
Generation of oxaloacetate occurs in the mitochondria
The enzymes that convert PEP to glucose are cytosolic
Either the oxaloacetate or PEP must leave the mitochondria
PEP is transported by specific transporter systems
Oxaloacetate transport is more complex as it can’t cross the mitochondrion membrane

Question 9

How is oxaloacetate transported out the mitochondrion membrane?

Answer 9

The malate aspartate shuttle
Oxaloacetate is converted into either malate (most common) or aspartate
Malate - uses malate dehydrogenase and NADH,, with the aid of a protein it can pass the membrane and turn back into oxaloacetate
Aspartate - uses aspartate aminotransferase, no NADH and a protein

Question 10

What is the second different step in gluconeogenesis?

Answer 10

The 7th step
Fructose-1,6-bisphosphate + H2O -> Fructose-6-phosphate + Pi
Uses Fructose bis-phosphatase

Question 11

What is the third different step in gluconeogenesis?

Answer 11

The 9th (final) step
Glucose-6-phosphate + H2O -> Glucose + Pi
Uses glucose-6-phosphatase

Question 12

What is the main regulatory point in gluconeogenesis?

Answer 12

Fructose bis-phosphatase - not a glycolytic intermediate but an allosteric activator

Fructose-2,6-bisphosphate activates PFK and inhibits FBPase, to maintain glycolysis when needed

Question 13

How else can glucoenogenesis be regulated?

Answer 13

Pyrvuate kinase can be allosterically inhibited by alanine, which is converted into pyruvate via transamination
This process increases gluconeogenic flux

Insulin inhibits the transcription of the gene for PEPCK
High concentrations of cAMP promote transcription of the gene for PEPCK

Question 14

Describe glycogen?

Answer 14

Glycogen is a polymer of a(1,4) linked D-glucose (chain length 8-14 residues) with a(1,6) linked branches every 4-8 residues
Glycogen occurs as intracellular granules (100-400 Å diameter), each contains ~ 120,000 glucose units (especially prominent in muscle and liver)
It is a storage polysaccharide, storing glucose to avoid osmotic stress

Question 15

What does the breakdown of glycogen involve?

Answer 15

3 enzymes
Glycogen phosphorylase
Glycogen debranching enzyme
Phosphoglucose mutase

Question 16

Glycogen breakdown - describe glycogen phosphorylase?

Answer 16

Cleaves glucose units from glycogen to yield G6P
It removes a glucose unit if it is at least 5 units away from branch point
Homodimer of 97 kDa subunits
It has a buried active site, which fits 4-5 sugars = processive enzyme
It has a covalently bound cofactor, PLP required for activity
PLP is a vitamin B6 derivative that is covalently linked via a Schiff base with a lysine residue
The phosphate of PLP acts as a general acid-base catalyst

Question 17

Glycogen breakdown - what is the mechanism of glycogen phosphorylase?

Answer 17

1. Formation of an E-P-glycogen ternary complex
2. Shielded oxonium ion intermediate formation from the alpha-linked terminal glucosyl residue involving acid catalysis by Pi, as facilitated by proton transfer from PLP. The oxonium ion has the half-chair formation 3. Reaction of Pi with the oxonium ion, forms glucose-1-phosphate

Question 18

Glycogen breakdown - what does glycogen phosphorylase do simply?

Answer 18

Catalyses glycogen phosphorolysis - bond cleavage by the substitution of a phosphate group
This yields glucose-1-phosphate

Question 19

Glycogen breakdown - how is glycogen phosphorylase regulated?

Answer 19

Regulated both by allosteric interactions and by covalent modification (phosphorylation and dephosphorylation)
Phosphorylation at a serine or threonine is one of the most common regulatory motifs in eukaryotes
Phosphorylase a = phosphorylated state
Phosphorylase b = unphosphorylated state
Allosteric inhibitors - ATP, G6P and glucose interact with the enzyme to change between R-state = active and T-state = inactive

Question 20

Glycogen breakdown - describe glycogen debranching enzyme?

Answer 20

Glycogen debranching enzyme is an a(1,4) transglycosidase which transfers an a(1,4) trisaccharide unit from a limit branch to the non-reducing end of a new branch
The remaining glucose unit can be hydrolysed by the same enzyme to yield glucose
This takes place due to glycogen phosphorylase only cleaves glucose units more than 5 from a branch point = the need for the removal of branch points
The enzyme has separate sites for the debranching (transferase) and hydrolysis (glucosidase) reactions
Independent active sites improves efficiency of the enzyme

Question 21

Glycogen breakdown - what does glycogen debranching enzyme do simply?

Answer 21

Removes glycogen’s branches

This makes additional glucose residues available to glycogen phosphorylase

Question 22

Glycogen breakdown - describe phosphoglucose mutase?

Answer 22

Phosphorylase generates G1P units which are converted to G6P by this enzyme
Major difference between this and the phosphoglycerate mutase of glycolysis is that this enzyme uses a Ser-P rather than a His-P

Question 23

Glycogen breakdown - what is the mechanism of phosphoglucose mutase?

Answer 23

Phosphorylation of the substrate at C6-OH

Phosphorylation of the enzyme by the phosphoryl group at C1 of the substrate

Question 24

Why is glycogen synthesis not a reversal of glycogen breakdown?

Answer 24

It is thermodynamically unfavourable without the input of energy

Question 25

What does glycogen synthesis involve?

Answer 25

3 enzymes:
UDP-glucose pyrophosphorylase
Glycogen synthase
Glycogen branching enzyme

Question 26

Glycogen synthesis - describe UDP-glucose pyrophosphorylase?

Answer 26

It activates glucosyl units to add to the glycogen chain
Combines G1P with uridine triphosphate to generated an “activated” compound (UDP-glucose)
UDP glucose can be added to the growing glycogen chain
This reaction is (deltaG) = 0, however coupled it is exergonic
This is a phosphoanhydride exchange reaction, this releases energy

Question 27

Glycogen synthesis - describe glycogen synthase?

Answer 27

Extends glycogen chains
Transfers glycosyl unit of UDP-G to the C4-OH of a non-reducing end of glycogen to form an a(1,4) glycosidic bond
Highly favourable (ΔG = -13.4 kJ/mol)

Question 28

Glycogen synthesis - how is glycogen synthase regulated?

Answer 28

Phosphorylation:
The dephospho form is more active and the phospho form is less active
Has multiple phosphorylation sites making more complex
Allosteric control
Inhibited by ATP, ADP and Pi at physiological concentrations
Almost totally inactive in vivo
Activated by G6P and allows rapid response to local needs

Question 29

Glycogen sythesis - describe glycogen branching enzyme?

Answer 29

Amylo-(1,4®1,6)-transglycosylase
Transfers a 7 glucose unit from the end of a chain to a C6-OH group of a glucose residue on the same or another glycogen chain

Question 30

Glycogen synthesis - what primes the glycogen synthesis?

Answer 30

Glycogenin
Acts as a glycosyltranferase, attches a glucose residue to the OH group of it’s Tyr 194
The extends the glucose chain by up to 7 donated glucose residues to form a glycogen primer
This allows glycogen synthase to bind

Question 31

Glycogen synthesis - what is the ideal structure of glycogen?

Answer 31

2 branch points, resulting in a roughly spherical molecule that is organised in tiers (a mature glycogen has roughly 12 tiers)
If it had more branches: the ends increase however so does particle density - which limits the size of the particle
Optimum chain length = 13 residues