Glycolysis and Glucose Oxidation Lecture - PDF

GLYCOLYSIS AND GLUCOSE OXIDATION Dr Nyree Myatt LEARNING OUTCOMES  Describe mechanisms of glucose uptake, indicating differences in types of transport and differences in cell context  Outline the potential metabolic fate of glucose-6-phosphate.  Outline the differences and similarities...

GLYCOLYSIS AND GLUCOSE OXIDATION Dr Nyree Myatt LEARNING OUTCOMES  Describe mechanisms of glucose uptake, indicating differences in types of transport and differences in cell context  Outline the potential metabolic fate of glucose-6-phosphate.  Outline the differences and similarities between glucokinase and hexokinase enzymes.  Describe the regulatory role of phosphofructo kinase enzyme in glycolysis.  Distinguish between the first 5 steps of glycolysis and the last 5 steps.  Describe how glucose generates ATP by substrate-level phosphorylation.  Relate glycolysis to the metabolism of fructose, glycerol and pentose sugars. TOPICS TO BE COVERED:  What is glucose?  Glucose uptake  Glycolysis  Interplay with other metabolic pathways  Glycerol (fatty acid metabolism)  Fructose metabolism  Pentose phosphate pathway  Learning objectives  GLUCOSE  Essential sugar for life! Most important source of energy.  Ringed molecule made up of 6 carbons (hexose).  Needs to be kept at a constant homeostatic level – controlled by hormones:  Too much – hyperglycaemic  Too little – hypoglycaemic (both serious conditions related to diabetes)  Can be stored as glycogen in liver and muscle  Most glucose-dependant organ? The brain  Normal range of glucose in blood?  Roughly 4 -6 mmol/l (70 – 100mg/dl) THE FATE OF GLUCOSE BUT FIRST! GLUCOSE UPTAKE  Before anything, glucose needs to be taken up into the cells  Issue: glucose is a charged (polar) molecule and cell membrane is hydrophobic.  Need specialized glucose transporters: GLUTs  facilitate transport across a concentration gradient (no ATP needed!)  14 different isoforms of GLUT – expressed in different tissues  Important GLUTs:  GLUT1: expressed in all cells (low Km), high in erythrocytes and blood-brain barrier endothelial cells  GLUT2: expressed in liver, small intestine and pancreas (high Km) – acts as a glucose sensor  GLUT 3: expressed in neurones and placenta  GLUT 4: expressed in muscle and adipose cells (low Km) – controlled by insulin.  GLUCOSE UPTAKE - GLUTS  14 different isoforms of GLUT – expressed in different tissues  Important GLUTs: GLUT 4 AND HORMONAL CONTROL  In skeletal muscles, most GLUT4 transporters are bound to vesicles inside the cell, so glucose cannot move inside.  Upon insulin signalling, these vesicle merge with the cell membrane, exposing the GLUT4 transporters and allowing glucose uptake.  Exercise also promotes this action (why exercise is good for diabetics)  GLYCOLYSIS  Simply: process which converts glucose to pyruvate.  10 step process split into 2 halves  First half requires energy (2 ATP) “investment”  Second half (“payback”) generates:  4 ATP  2 NADH (energy rich molecule)  2 pyruvate molecules (used in Krebs cycle to generate much more ATP)  1: PHOSPHORYLATION OF GLUCOSE  First step is to add a phosphate group to glucose, forming glucose -6 – phosphate (G6P).  Requires one ATP (energy draining step)  This is required to prevent glucose from leaving the cell  Catalysed by the enzyme hexokinase  can phosphorylate any 6 ringed carbohydrates  Expressed in mostly all cells  Sensitive to negative feedback from G6P.  Knock on effect on glucose uptake by GLUT.  Only found in liver and pancreas  Catalytically prefers glucose. Why?  Responds quicker to changes in glucose levels  High Km STEP 1 No feedback inhibition – it will keep working even in high ALTERNATIVE:  glucose environments  Acts as a sensor for glucose levels in β-cells of the GLUCOKINASE pancreas to regulate insulin/glucagon production/secretion  Glucokinase regulation Activity of glucokinase is regulated by localisation by binding it to the protein glucokinase – regulatory protein (GKRP). Low glucose concs – GKRP stays in nucleus bound to glucokinase At higher glucose concs, glucokinase dissociates from regulatory protein In liver, Glucokinase shuttles glucose into producing glycogen Glucokinase better at responding to differing glucose concs than hexokinase  STEP 2: ISOMERIZATION  Changing G6P into one of its isomers: fructose – 6- phosphate  Makes later chemical reactions more energetically favourable STEP 3: PHOSPHORYLATION OF FRUCTOSE 6-PHOSPHATE  Important step: rate- limiting step by regulating the function of the enzyme phosphofructokinase 1  Adds another phosphate group (uses another ATP - down 2 ATPs) CONTROL OF PHOSPHOFRUCTOKINASE 1  Phosphofructokinase 1 (PFK1) enzyme can be regulated by a number of molecules which will determine whether glycolysis moves forward, stops or &ADP backwards.  PFK1 is allosterically activated by:  High AMP/ADP levels (cell needs energy/ATP)  Fructose-2,6-bisphosphate (F2,6P) produced from fructose 6-phosphate by an isoform of PFK1, called PFK2, an enzyme which is hormonally regulated.  PK1 is inhibited by products of glycolysis and the Krebs cycle:  High ATP levels  Citrate (a byproduct of the Krebs cycle)  H+ - protects muscle from damage if too much acid accumulates Phosphofructokinase -2 is a bifunctional enzyme – it has kinase and phosphatase activities. Insulin stimulates the kinase activity leading to fructose 2, 6 bisphosphate (stimulates PFK-1). Glucagon stimulates the phosphatase activity and gluconeogenesis  Question time Which GLUT acts as a glucose sensor? Which enzymes can phosphorylate glucose? Which enzyme is the first dedicated to glycolysis rate limiting one? What inhibits this enzyme? STEP 4: BREAKDOWN OF 6 CARBON RING TO TWO - 3 CARBON CHAINS  Fructose 1,6-bisphosphate is broken down the middle to give two 3 carbon chains, both carrying one phosphate group but with different structures: like non-identical twins!  One is an aldehyde – glyceraldehyde-3-phosphate  Other is an acetone – dihydroxyacetone phosphate  Catalysed by the enzyme aldolase.  The acetone can be converted to the aldehyde through an isomerisation reaction (step 5), since only the aldehyde can be used for glycolysis.  So now we have two identical glyceraldehyde – 3 – phosphate molecules from one glucose.  How many ATP’s are we at now? (+ or -) STEP 6: ADDING AN INORGANIC PHOSPHATE  Prior to generating our first ATP, we need to add a phosphate group from the cytosol (rather than from ATP as was happening before)  The dehydrogenase enzyme catalyses the addition of a free phosphate to the glyceraldehyde producing a 3 carbon chain carrying 2 phosphate groups (a high energy molecule)  As a by-product of this reaction, NAD+ is reduced to NADH plus a free proton (H+). This is super important!  NADH is used later in the KREB’s cycle and electron transport chain to regenerate NAD+ which is needed to keep glycolysis going.  NAD+ can also be regenerated by lactic acid produced from anaerobic breakdown of glucose. STEP 7: FIRST ATP PRODUCED  The high energy molecule 1,3-bisphosphoglycerate gives one of its phosphates to generate one ATP.  Catalysed by phosphoglycerate kinase.  Substrate level phosphorylation: means that we are just shifting phosphate groups around to create ATP (different from how ATP is generated later in the electron transport chain)  So what is our ATP count at this step? STEP 8: SHIFTING THE PHOSPHATE  Phosphate group shifted from one carbon to the middle carbon – catalysed by phosphoglyceromutase. (NB: 2,3- diphosphoglycerate – recognise this from O2 dissociation curve?) STEP 9: DEHYDRATION  Removal of a water molecule produces phosphoenolpyruvate (PEP)  PEP is an important intermediary since it can also be produced by gluconeogenesis.  Enzyme is enolase (can be inhibited by fluoride ions – toothpaste and some blood test vials so glucose concs can be determined )  STEP 10: ANOTHER ATP IS PRODUCED!  The last step of glycolysis produces the last ATP from PEP by the enzyme pyruvate kinase.  The production of pyruvate is also important since it feeds into the next energy producing cycle – the Kreb’s cycle.  This also is a substrate level phosphorylation of ATP from ADP.  So what is the NET production or loss of energy of glycolysis?  2 pyruvates (so still have 6 carbons), 2 NADH, 4ATPs (net + 2ATPs) Quick recap – take a moment to answer the following What is glycolysis? How does glucose get into cells? How is glucose kept in cells? What happens to dihydroxyacetone phosphate What is substrate level phosphorylation? How many reactions in glycolysis are irreversible?  SUMMARY  Video  Key points to remember:  1st 5 steps are energy depleting  2nd 5 steps are energy generating  In all 2 ATPS are consumed and 4 generated  2 pyruvate molecules are also generated per glucose  And 2 NADH molecules  Most important step is regulation is step 3, catalysed by phosphofructo kinase 1 enzyme – a rate limiting reaction.  Aerobic versus anaerobic If oxygen is present, pyruvate is catalysed to acetyl CoA by pyruvate dehydrogenase AcetylCoA then reacts with oxaloacetate in the Krebs cycle If oxygen is scarce, glycolysis could stop if NAD+ is not regenerated from NADH Atthis point, pyruvate can act as the electron acceptor and be reduced to lactate, and NADH is re-oxidised to NAD+ Enzyme is lactate dehydrogenase Lactatetravels to liver, converted to pyruvate – Cori Cycle – and then to glucose (gluconeogenesis) WHAT HAPPENS TO PYRUVATE? INTERACTION WITH GLYCEROL (FATTY ACID METABOLISM)  Glycerol is converted into dihydroxyacetone phosphate (DHAP) which can feed into glycolysis at step 4.  Glycerol is converted to glycerol -3-phosphate by glycerol kinase (mainly in liver), which in turn is oxidised to DHAP, releasing another NADH (glycerol-3-phosphate dehydrogenase) INTERACTION WITH FRUCTOSE METABOLISM  Fructose, like glucose, is a 6-carbon hexose carbohydrate  Very common in the diet (common sugar)  Can be metabolised by hexokinase to fructose 6–phosphate and joins glycolysis at step 3 (occurs in all cells)  Alternatively, liver produces fructokinase (kinase that acts only on fructose) to produce fructose-1-phosphate. This can be converted immediately to dihydroxyacetone phosphate and glyceraldehyde by the enzyme aldolase B to join glycolysis at step 5.  Biochemistry in the news – fructose is postulated to drive obesity and colorectal cancer: fructose survival switch  https://www.news-medical.net/news/20241205/Liver-conversion-of-fructose-fuels-canc er-growth-by-supplying-lipids-for-tumor-proliferation.aspx  https://www.nih.gov/news-events/nih-research-matters/how-fructose-may-contribute- obesity-cancer FRUCTOSE IN GLYCOLYSIS Essential hepatic fructosuria: Autosomal recessive genetic condition caused by mutations in KHK gene (ketohexokinase gene). Causes deficiency in fructokinase enzyme leading to patients being unable to process fructose. Can be controlled by avoiding sugary foods. Only in LIVER Hereditary fructose intolerance is caused by a deficiency in aldolase B enzyme (– can kill liver cells) INTERACTION WITH: PENTOSE PHOSPHATE PATHWAY  An important pathway for the production of 5- carbon sugars (pentoses) used in nucleic acids (that make up DNA and RNA) and NADPH needed for steroid hormones and fatty acids synthesis.  Important in liver and red blood cells.  Important products of pentose pathway:  Ribose 5 phosphate  NADPH (different from NADH)  Pentose pathway also produces glyceraldehyde-3-phosphate and fructose-6- phosphate, both of which can feed into glycolysis.  How many ATPs are needed in glycolysis and how many are produced?  Why is NAD important and how is it produced when oxygen is scarce?  Why is PFK1 important and how is it regulated?  How are triglycerides linked to glycolysis? QUESTION TIME  2 ATPs needed in the “investment” phase and 4 are produced in the ”pay back“ phase  NAD is needed to oxidise glyceraldehyde 3 phosphate to 1,3 bisphosphateglycerate via glyceraldehyde 3 phosphate dehydrogenase. NAD is reduced to NADH in this step. Without NAD, this step can’t take place and glycolysis stops. Pyruvate steps in as the electron acceptor from NADH and forms lactate and NAD when oxygen is low.  PFK1 is the committed step in glycolysis. It senses the energy charge in the cell. It is regulated by ATP.ADP and AMP. It is inhibited by ATP, but upregulated by ADP, AMP and fructose 2,6 bisphosphate which is produced by PFK2 from fructose 6 phosphate.  Glycerol is produced from the degradation of triglycerides. Glycerol is fed into glycolysis via glycerol 3 phosphate (via glycerol kinase) and then dihydroxyacetone phosphate which is produced via glycerol 3 phosphate dehydrogenase and NAD ANSWERS ALL QUESTIONS CORRECT? GOLD STAR! LEARNING OUTCOMES  Describe mechanisms of glucose uptake, indicating differences in types of transport and differences in cell context  Outline the potential metabolic fate of glucose-6-phosphate.  Outline the differences and similarities between glucokinase and hexokinase enzymes.  Describe the regulatory role of phosphofructo kinase enzyme in glycolysis.  Distinguish between the first 5 steps of glycolysis and the last 5 steps.  Describe how glucose generates ATP by substrate-level phosphorylation.  Relate glycolysis to the metabolism of fructose, glycerol and pentose sugars.