Term 2 Lecture 10 : Glycolytic Pathway Flashcards
Process
Glucose + 2NAD+ 2ADP
→2NADH+2ATP+2 Pyruvate
- doesn’t produce much ATP however anaerobic organisms rely on this method to get ATP for life
- glucose passes through 10 enzyme catalysed reactions to become Pyruvate
- neg free energy change, so reaction is favoured and therefore spontaneous
10 steps
1) hexokinase hydrolyses an ATP→ADP
Puts a pyruvate group on a glucose molecule turning it into glucose 6 phosphate
2) glucose 6 phosphate isomerase turns glucose 6 phosphate into fructose 6 phosphate
3) 6 phospofructose kinase hydrolyses an ATP→ADP and adds a pyruvate group converting fructose 6 phosphate to fructose 1,6 biphosphate
4) fructose biphosphate aldylase splits fructose 1,6 biphosphate into dihydroxyacteone phosphate and glycerol aldehyde 3 phosphate
(2 x 3 carbon molecules)
5) triose phosphate isomerase converts dihydroxyacteone phosphate to glyceraldehyde 3 phosphate (so now there are 2 of the same molecule)
6) glyceraldehyde 3 phosphate dehydrogenase uses 2NAD+ to remove a H from each of the 2 molecules to oxidise 2NAD+ to 2NADH releasing 2 phosphates leaving 2 molecules of 1,3 biphosphoglycerate
7) phosphoglycerate kinase removes one phosphate from each molecule (by converting 2ADP→2ATP) making 2 molecules of 3- phosphoglycerate
8) phosphoglycerate mutase converts the 2 molecules from 3-phosphoglycerate to 2 phosphoglycerate
9) phosphopyruvate hydrolase removes a water molecule from each molecule leaving 2 phosphoenolpyruvate
10) finally 2ADP→2ATP by pyruvate kinase producing 2 ATP overall and resulting in 2 Pyruvate molecules.
Basically 2 ATP are used (steps 1 & 3) to produce fructose 1,6 biphosphate and 4 ATP are produced by phosphorylation of ADP in steps 7 & 10
Glucose + 2 Pi+2 ADP + 2NAD+
→ 2 pyruvat+ 2ATP+2NADH+2H+
∆G⁰’ = -197kjmol-¹
Issues with glycosylation
The combustion of glucose results in ∆G⁰’= -2870kjmol-¹ so a lot of energy is left unreleased
Accumulation of NADH also causes a problem (this is why it’s converted to lactate) it leads to inhibition of glyceraldehyde -3- phosphate dehydrogenase (necessary for step 6)
ATP production by phosphorylation during glycolysis is sometimes referred to as “substrate level phosphorylation”
Steps 1 & 3 have a large neg ∆G so must be controlled - control at these irreversible steps must be near constant for all other metabolic processes to function correctly.
Control of glycolysis
Conversion of glucose to fructose 6 phosphate (steps 1&2) minor control by product inhibition - as product blocks the active site of hexokinase
Conversion of fructose 6 phosphate to fructose 1,6 phosphate (step 3) is a major control point. ATP, citrate and H+ negatively regulate the process (slow it down) whilst AMP (adenosine monophosphate) and fructose 2,6 biphosphate positively regulate it
Conversion of phosphoenolpyruvate to pyruvate (step 10) is also regulated. Positive regulator is fructose 1,6 biphosphate and negative regulators are ATP and alanine ( preventing glycolysis from overriding biosynthetic processes)
Phosphofructokinase
A tetrameric enzyme with 4 catalytic sites and 4 allosteric sites (which give forward inhibition)
Fructose 6 phosphate+ ATP
→ADP+fructose 1,6 biphosphate
At low ATP a Michaelis Menten curve
At high ATP a sigmoidal curve
This is due to ATP filling the allosteric sites as well as catalytic sites slowing the reaction down
> ADP and AMP also interact to activate the enzyme
Enzyme is inhibited by citrate derived from pyruvate (end product of glycolysis) and H+ (feedback inhibition)
Fructose 2,6 biphosphate binds to allosteric sites on the enzyme and stops ATP from entering so it cannot slow the reaction - enhancing activity
Removal of NADH
In most aerobic organisms it is removed by oxidative phosphorylation in anaerobic organisms other pathways are used
TCA cycle - what happens to the pyruvate produced by glycolysis
In aerobic microorganisms and mitochondria pyruvate enters the TCA cycle and is combined with O2 in a process known as complete oxidation
Ethanol →CO2 + H2O
This process produces lots of ATP
Anaerobic processing of pyruvate
Anaerobic fermentation
Pyruvate to ethanol
1) H+ is taken up to form pyruvate decarboxylase then CO2 is released
2) Acetaldehyde is formed
3) NADH + H+ is taken in forming alcohol dehydrogenase then NAD+ is released
4) ethanol is formed
E.g. alcoholic fermentation in yeast
Glucose+2Pi+2ADP →2 ethanol+2CO2 + 2ATP
Anaerobic metabolism
Pyruvate to lactate e.g. in mammalian muscle buildup leads to cramping
Pyruvate+H+ + NADH →lactate + NAD+
Homolactic fermentation occurs in animal cells and lactic acid bacteria
To release more energy from pyruvate
Pyruvate + Co A →pyruvate dehydrogenase→Acetyl Co A
Pyruvate dehydrogenase is one of the largest enzymes found in any organism with a molecular weight of 4-10 million Dalton’s it contains 3 enzyme reactions and uses 5 cofactors (aka coenzymes)
It is visible by electron microscopy being 20-40nm across
3 parts:
E1: pyruvate dehydrogenase (24)
E2: dihydrolypyl transacetylase (24)
E3: dihydrolypyl dehydrogenase (12)
24+24+12 = 60 subunits
How pyruvate dehydrogenase works
E1: converts pyruvate to hydroxyalthyl thiaminediphosphate (using cofactor thiamine diphosphate) releasing CO2
E2: breaks disulphide bond and removes acetyl group from hydroxyacetyl thiaminediphosphate and attaches a lipo amide chain forming acetyl lipo amide to which Co A is added
Acetyl lipo amide+ Co A →acetyl Co A + dihydroxylamide
E3 regenerates lipoamidr by oxygenation and reduction of NAD+ and FADH
What is Co A (Aka CoASH)
- acetyl group from pyruvate is attached to carrier molecule Co A to generate acetyl Co A an activated intermediate
- Co A structure provides a “handle” which binds with high affinity to metabolic enzymes
- reactive part is a sulphydryl group which forms a thioester linkage with carboxyl groups
Sources of Acetyl Co A
Pyruvate+ Co A + NAD+
→Acetyl Co A+CO2+NADH
∆G⁰’ = -33.5kjmol-¹
Other sources of Acetyl Co A - fatty acid oxidation - catabolism of fatty acids from dietary lipids by hydrolysis - major energy source for humans, more ATP is generated from fats than sugars
Most catabolism is based on fatty acids derived from intracellular stores of triacylglycerol fats.
It takes place in the mitochondria and generates reduced cofactors that can produce ATP via oxidative phosphorylation
Fatty acid oxidation can also take place in peroxisomes where no reduced cofactor is generated and energy is dissipated as heat
Free fatty acids are toxic in large amounts so for transport are reconverted to trialglycerols or bound to carriers
Fatty acid toxicity is particularly a problem in type 2 diabetes associated with obesity
Intracellular hydrolysis of trialglycerides is carried out by lipases regulated by hormones (via signal transduction systems)
Acetyl Co A is fed into the TCA cycle (AKA Krebs or citric acid cycle)
See diagram start of notebook 3
TCA cycle takes in two-carbon units as acetyl-Co A and oxidises them to CO2 generating energy in the form of reduced cofactors (NADH and FADH2) and some GTP (convertible to ATP) is also produced
The cycle does not use O2 but provides reduced cofactors to drive oxidative phosphorylation - where O2 reduction to H2O drives ATP production.
TCA cycle also produces precursors for biosynthetic processes leading to fatty acids etc.
TCA cycle equation
Acetyl CoA +3NAD++FAD+ GDP+Pi+2H2O
↓
2CO2+3NADH+FADH2+GTP+2H+ + CoA
Reactions in the TCA cycle generally have small ∆G⁰’ values (except malate dehydrogenase) and are reversible in vivo
TCA cycle steps
Step 1)
C4+C2→C6 catalysed by citrate synthase
Oxaloacetate+ acetyl CoA
→ citryl CoA+ citrate
Condensation followed by hydrolysis to give CoA
Step 2)
C6→C5+CO2 first isomerisation catalysed by aconitase
Citrate →isocitrate
Hydroxyl group is moved to correct position for oxidative phosphorylation then oxidative decarboxylation is catalysed by isocitrate dehydrogenase
Isocitrate+NAD+
→oxalosuccinate+NADH + H+
→alpha ketoglutarate+ CO2
Step 3)
C5→C4+CO2 oxidative decarboxylation catalysed by alpha ketoglutarate dehydrogenase compound
Alpha ketoglutarate+ NAD+ + Co A
→succinyl Co A
Step 4)
GTP formation catalysed by succinyl Co A synthetase
Succinyl CoA+ Pi + GDP
→ succinate + Co A + GTP
Step 5) regeneration of oxaloacetate
Succinate + FAD→fumarate+FADH2
Catalysed by succinate dehydrogenase
Fumarate+H2O→malate
Catalysed by fumarase
Malate+NAD+→oxaloacetate+NADH+H+
Catalysed by malate dehydrogenase
