Metabolism - Block 5 Flashcards
Is Glycolysis + Krebs Cycle an anabolic or catabolic process?
Catabolic: sugar»_space; CO2
Are protein, fat & polysaccharide synthesis anabolic or catabolic processes?
Anabolic: building molecules
Is ATP made or used up during catabolism?
ATP is made
Is ATP made or used up during anabolism?
ATP is used up
Is NAD(P)H made or used up during catabolism?
NAD(P)H is made
Is NAD(P)H made or used up during anabolism?
NAD(P)H is used up
NADPH is “nature’s favourite” what?
Nature’s favourite reducing agent
What is often used as a model for NADPH, and why?
DCPIP is used instead of NADPH
This is because:
1. it is stable, unlike NADPH, and
2. it is blue, so the blue»_space;> colourless reaction can be seen, unlike with colourless NADPH
Where in the cell does glycolysis take place?
The cytosol
Where in the cell does the Krebs cycle take place?
The mitochondrion
What occurs in the first step of glycolysis?
Glucose is converted (phosphorylated) to G6P
What happens to G6P in glycolysis (step 2)?
G6P is converted to F6P (isomerisation)
Which enzyme converts glucose to G6P?
Hexokinase
Which enzyme converts G6P to F6P?
Phospho-hexose isomerase
What happens to F6P in glycolysis (step 3)?
F6P is converted to F(1,6)BP (isomerisation)
Which enzyme converts F6P to F(1,6)BP?
Phosphofructokinase
What happens to F(1,6)BP in glycolysis (step 4)?
It is split into GAP and DHAP (3-Carbon molecules)
Which enzyme converts F(1,6)BP to GAP and DHAP?
Aldolase
What happens to GAP and DHAP in glycolysis (step 5)?
DHAP is converted to GAP, so two GAP are present
What happens to the two GAP in glycolysis?
They are oxidised to GBP (a carboxilic acid with two phosphates)
What oxidant is needed to convert GAP to G(1,3)BP?
NAD+
What important process occurs when G(1,3)BP is converted to G3P?
The formation of (2) ATP (as there are two GBP molecules to begin with)
What happens to G3P in glycolysis?
It is isomerised to G2P
Which steps in glycolysis use ATP?
- The phosphorylation of glucose to G6P
2. The phosphorylation of F6P to F(1,6)BP
Which steps in glycolysis make ATP?
- The dephosphorylation of G(1,3)BP to G3P (2x ATP made)
2. The conversion of PEP to enolpyruvate (followed by pyruvate) in the final step
Which steps in glycolysis are irreversible?
The ones which use ATP (phosphorylations) AND the final step:
- Conversion of glucose > G6P (first step)
- The phosphorylation of F6P to F(1,6)BP
- Conversion of PEP to (first enolpyruvate, then) Pyruvate
What happens to G2P in glycolysis (penultimate step)?
A lyase splits out H2O and converts it to PEP (phospho/enol/pyruvate)
What happens to PEP in glycolysis (final step)?
It is dephosphorylated by pyruvate kinase (making 2x ATP) into enolpyruvate, which converts by itself to Pyruvate
What is the net production of ATP after glycolysis?
+2 ATP
What does coenzyme A act as?
A handle for carrying acid groups
What happens to Pyruvate (3C) in the Krebs Cycle (step 1)?
Its CO2 group is swapped for CoA, forming Acetyl CoA (C2).
It loses H atoms to NAD+, making NADH + H.
What happens to Acetyl CoA (2C) in the Krebs Cycle (step 2)?
It reacts with Oxaloacetate (C4) to form Citrate (C6)
What happens to Citrate (C6) in the Krebs Cycle (step 3)?
It is converted to Isocitrate (C6) then 2-Oxo-glutarate (5C)
A CO2 is lost,
It also loses H atoms to NAD+, making NADH + H.
What happens to 2-Oxo-glutarate (5C) in the Krebs Cycle (step 4)?
It is converted to Succinyl-CoA (4C)
A CO2 is lost,
It also loses H atoms to NAD+, making NADH + H.
What happens to Succinyl-CoA (4C) in the Krebs Cycle (step 5, beginning of 4C home straight)?
CoA is removed to convert it to Succinate (C4) and the free energy associated with the reaction makes GTP
What happens to Succinate (4C) in the Krebs Cycle?
It is dehydrogenated to Fumarate (4C),
Hydrogens taken up by FAD to make FADH2,
This results in an unsaturated bond forming.
What happens to Fumarate (4C) in the Krebs Cycle?
It undergoes a lyase reaction,
Unsaturated bond replaced by an OH and a H,
It forms Malate as a result.
What happens to Malate (4C) in the Krebs Cycle?
It is oxidised to Oxaloacetate (C4),
It also loses H atoms to NAD+, making NADH + H.
What does NAD+ stand for?
Nicotinamide adenine dinucleotide
What two nucleotides are present in NAD+?
Adenine and nicotinamide (Vit B3)
Where does the extra P join onto NAD+ to make it NADP?
Onto the ribose of the adenine
Where is the the + charge on the NAD+ molecule?
The sugar-base bond N on the nicotinamide base
What source might provide H atoms for reduction of NAD+ to NAD(P)H?
A secondary alcohol CHOH group
What kind of pathways are NAD+ and NADH usually involved in?
Catabolic pathways (making ATP)
What kind of pathways are NADP+ and NADPH usually involved in?
Anabolic pathways (biosynthesis)
How can you distinguish experimentally between NAD(P)+ and NAD(P)H?
UV Spectrophotometry
The oxidised forms, NAD(P)+, do NOT absorb UV
The reduced forms, NAD(P)H, DO absorb UV
What kind of enzyme usually creates NADH?
Dehydrogenases (in glycolysis and KC)
What is the exception to the idea of dehydrogenases creating NADH?
Succinate dehydrogenase (succinate >>> fumarate) creates FADH2 instead of NADH, This is because succinate is too weak of a reducing agent to make NADH.
How many molecules of NADH are made per glucose in glycolysis + KC?
10 NADH per glucose
How many molecules of FADH2 are made per glucose in glycolysis + KC?
2 FAD2 per glucose
What process is the source of NADPH?
The oxidative pentose phosphate shunt
What is the yield of the oxidative pentose phosphate shunt?
2x NADPH
1x Ribose-5-phosphate
What happens to excess Ribose-5-phosphate (5C) from the oxidative pentose phosphate shunt?
It is converted into F6P (6C) and G3P (3C) and used in the glycolysis pathway.
What happens to excess NADPH from the oxidative pentose phosphate shunt?
F6P and G3P molecules will be drawn from the glycolysis pathway and into the complex sugar interconversion pathway, and Ribose-5-phosphate is produced.
Where in the cell doesthe oxidative pentose phosphate shunt take place?
In the cytosol (like glycolysis)
What cation is ATP normally complexed with?
Mg^(2+)
How can ATP be replaced?
- Substrate-level phosphorylations (glycolysis and KC)
2. Oxidative phosphorylation - buildup of NAH+ forms pH gradient and drives ATP production
Where does oxidative phosphorylation take place?
The cristae of the mitochondrion
Which produces more ATP: oxidation of 1x NAHD or oxidation of 1x FADH2?
NADH oxidation produces more ATP
> about 2.5 ATP
> about 1.5 ATP is produced by FADH2 oxidation
What important group is present on a CoA molecule?
An SH (thiol, sulfhydryl) group
How does CoA “carry” acids?
Acids join to the S atom via a thioester bond
How can starch be used as fuel for catabolism EXTRACELLULARLY?
Since starch is a polymer of glucose, it can by hydrolysed by hydrolytic enzymes to the monosaccharide.
How can starch be used as fuel for catabolism INTRACELLULARLY?
Since starch is a polymer of glucose, phosphorylase enzyme attacks the polymer forming glucose-1-phosphate.
Why is it more benificial to use a phosphorylase enzyme (intracellular) than a hydrolytic enzymes (extracellular) when catabolising starch?
> Hydrolytic enzymes converts starch to glucose, where it can enter the glycolysis pathway.
> Phosphorylase converts starch to glucose-1-phosphate (G1P), an isomer of G6P. With the help of a mutase, this means the first step of glycolysis can be bypassed and one fewer ATP is spent.
How can inulin be catabolised?
> As a fructose polymer, it can be hydrolysed to fructose.
> A fructokinase can convert this to F6P (with the help of ATP) where it can fit into the glycolysis pathway.
How can pectin (dietary fibre) be catabolised?
> As a polysaccharide, it can be hydrolysed to many sugars, one of which is L-Arabinose (5C).
> With an isomerase, a kinase and an epimerase (and one ATP) L-Arabinose can be converted to D-Xylulose, which can be ustilised in the Pentose Phosphate Shunt.
How can benzene be catabolised?
> Benzene is first oxidised (Both O addition and H removal) so it loses aromaticity.
> Some isomerisations then occur,
> The intermediate is condensed with CoA,
> This intermediate is hydrolysed into two products: Acetyl-CoA (C2) and succinate (C4).
How can excess amino acids be catabolised?
- The amino group is removed by transamination with an oxo-acid (usually 2-oxoglutarate from KC).
- The amino acid is converted to a new oxo-acid, and the 2-oxoglutarate is converted into glutamate.
- The glutamate is dehydrogenated back into 2-oxoglutarate, which is used in another transamination or in the KC. NH3 is also produced here, and is excreted.
- The new oxo-acid is reacted with a series of enzymes similar to those at the final steps of KC, cleaved with CoA, and then finally converted into Succinyl-CoA and Acetyl-CoA.
How can fats be catabolised?
- The three ester bonds of the fat are hydrolysed by esterases, forming glycerol and 3x fatty acids.
- Glycerol is phosphorylated by ATP, and then oxidised, forming NADH + H, to finally produce DHAP.
- The fatty acids are reacted with CoA as acids are wont to do, with ATP being converted to AMP in the process.
4a. The fatty-CoA undergoes a C-C to C=C reaction (producing FADH2), similar to a Succtinate to Fumarate conversion.
4b. The new intermediate undergoes a hydration, similar to a Fumarate to Malate conversion.
4c. The new new intermediate undergoes a dehydrogenation (producing NADH + H), similar to a Malate to Oxaloacetate conversion.
4d. This intermediate is cleaved with CoA, similar to AA catabolism (thiolytic cleavage) and forms Acetyl-CoA plus a shorter fatty-CoA
- The shorter fatty-CoA (1C shorter) goes back to the beginning of step 4 to be shortened all over again (( β-Oxidation Spiral )).
What are two alternative ways to make ATP?
> Oxidative phosphorylation
> Photophopsphorylation
What happens to G3P in photosynthesis?
What are the three differences between photosynthesis and glycolysis with respect to this?
> G3P is phosphorylated to 1,3-BPG, using ATP
The 1,3-BPG is then converted to GAP
> This is the opposite direction to glycolysis
ATP is used instead of made
NADPH is the carrier instead of NADH
What does anabolism require?
> ATP
> NADPH
What is the plant equivalent of the mitochondrial matrix?
The stroma of the chloroplast.
What does the Reductive Pentose Phosphate Pathway produce from the 6x G3P?
> A Profit of 1x G3P is made and used for anabolism
> 3x ATP are produced to go back into the pathway (which uses x6 ATP to make G3P)
Outline how a metabolic pathway could be traced with radiolabelling.
> Label a certain substance with a radioisotope, e.g. [C14] Ascorbate, and feed it into the cells.
> Kill the cells at a set time.
> Analyse the cells with chromatography or electrophoresis.
> Detect, quantify & ID the radioactive spots.
What are some ways you could kill cells quickly at a set time to investigate a snapshot of their metabolism?
> Ethanol
> Liquid N2
> Formic acid
How can the radioactive spots be detected?
Autoradiography?
How can the radioactive spots be quantified?
A Geiger or scintillation counter.
What are the two methods of radioactive labelling?
- Pulse-chase - The radioactive precursor is quickly used up, and is traced directly.
- Continuous - The supply of added radioactive substance is inexhaustible, and radioactivities of all components reach a plateau (except the final product, which is exponential).
