Lecture 19: Glycolysis: A Starting Point of Metabolism - PDF

Glycolysis: A starting point of metabolism 4BICH001W Biochemistry Dr Sarah K Coleman Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asy...

Glycolysis: A starting point of metabolism 4BICH001W Biochemistry Dr Sarah K Coleman Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). METABOLIC PATHWAYS… What part do you not understand? Navigating metabolic pathways…  General Organisation A pathway: a sequence of reactions. – Many small changes to cause a large significant change – Location matters: In brain or skeletal muscle or gut in cytosol or mitochondria or nucleus – Most pathways are: Some pathways are:  Glycolysis The glucose breakdown pathway Key steps What happens next Some core ideas for metabolic pathways  General Overview Molecules are broken down in stages (not just glucose): Stage 1 Large molecules are broken down to smaller ones. e.g. polysaccharides are broken down to simple sugars. Stage 2 Small molecules are broken down to simpler compounds that play a key role. The major end product is Acetyl-CoA  General Overview Stage 3 The 2 carbon fragment of Acetyl CoA is oxidised in the TCA cycle (Krebs cycle) During this process… - electrons are released from oxidation of acetyl group - these electrons transferred via carriers (e.g. NADH) to the inner mitochondrial membrane. - Electrons passed down the electron transport chain to the final electron acceptor oxygen. Oxygen is reduced to water. - This process will drive the generation of ATP (oxidative phosphorylation). Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Glycolysis Glucose is the major fuel of most organisms and occupies a central role in metabolism. Rich in potential energy - complete oxidation of glucose to carbon dioxide and water has Go’ = -2840 kJ/mol. (Standard free energy change)  Glycolysis By storing glucose as a high molecular weight polymer (starch or glycogen) a cell can stockpile large quantities of hexose units while maintaining a relatively low cytosolic osmolarity. When energy demands suddenly increase, glucose can be rapidly released from these storage polymers to produce ATP.  Glycolysis, the TCA cycle and electron transport Remember: Metabolic pathways do not exist in Krebs cycle is also isolation called Citric Acid cycle Intermediates or Tricarboxylic Acid and products cycle (TCA) feed into each other  Triacyl glycerols Polysaccharides sugars Proteins Glycerol Free fatty acids Beta oxidation Amino acid skeletons  Glycolysis Derived from the Greek ‘glykys’ meaning sweet and ‘lysis’ meaning splitting. Glycolysis an (almost) universal central pathway of glucose catabolism. Sole source of metabolic energy in some tissues eg in brain, erythrocytes and some anaerobic microbes. First metabolic pathway to be elucidated. 10 reaction steps. Overall schema: Glucose + 2 NAD+ + 2 ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATP + 2H2O + 2 H+  Glycolysis A set of sequential enzyme reactions breaks down glucose to two 3- carbon (C) compounds. Some of released free energy conserved in the form of ATP and NADH. There are two parts to the pathway: – Phase I (Reaction steps 1-5): free energy content of intermediates is raised using ATP. – Phase II (reaction steps 6-10): the pay-off phase (energy is liberated)  Glucose Found in blood from breakdown of polysaccharides e.g. glycogen, starch Enters cell cytosol by specific transporter proteins Enzymes for glycolysis are located in cytosol Other hexose sugars e.g. fructose, mannose, galactose, get converted into intermediates of the glycolytic pathway.  Glycolysis Energy generation (production) is incorrect Glycolysis generates ATP This is energy transduction  Glycolysis In summary 1) The 6C sugar Glucose is broken down to two 3C compounds (pyruvate) 2) 2x ATP are used up early on, 4x ATP are generated in the later phases. (net gain 2) 3) 2 molecules of NADH are formed (ie reducing power) 4) 3 steps are essentially irreversible, the majority are reversible 5) ADP, ATP, NAD+ and inorganic phosphate Pi are required for glycolysis to proceed 6) The pathway is a source of biosynthetic intermediates Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Control of Glycolytic Pathway 3 steps are essentially irreversible, the majority are reversible Irreversible steps = key control points Reactions will have large negative ΔG changes under physiological conditions Control points due to the enzymes involved and how regulated: – Hexokinase (step 1) – Phosphofructokinase (step 3) – Pyruvate kinase (step 10) We will focus on the ‘control’ enzymes and reaction steps  Hexokinase Glucose + ATP Glucose-6-phosphate + ADP Reaction One of Glycolysis: The reaction is essentially irreversible; a key control point  Hexokinase Glucose + ATP Glucose-6-phosphate + ADP Hexokinase is an enzyme which will phosphorylate hexose sugars. Glucose is the preferred substrate; but it can phosphorylate other hexose sugars e.g. fructose and mannose. Kinase enzymes add a phosphate group (comes from ATP, adenosine triphosphate). Muscle hexokinase is allosterically inhibited by its product glucose-6- phosphate (G6P); making it an important control point. – An allosteric inhibitor binds at a site other than the active site.  Isoenzymes: Hexokinase and Glucokinase Glucose + ATP Glucose-6-phosphate + ADP Liver cells (hepatocytes) contain a form of hexokinase called glucokinase. Glucokinase is more specific for glucose and differs in its regulation from hexokinase. It is an isoenzyme (a different protein that carries out the same reaction) of hexokinase Why the need for the two? - Muscle and liver have different roles; muscle consumes glucose, liver produces it. Glucokinase Km = 10 mM; Hexokinase Km = 0.1 mM  Phosphofructokinase Fructose-6-phosphate + ATP Fructose-1,6-bisphosphate + ADP Reaction Three of Glycolysis: The reaction is essentially irreversible; a key control point  Phosphofructokinase Fructose-6-phosphate + ATP Fructose-1,6-bisphosphate + ADP Phosphofructokinase (PFK) converts fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F1,6P or FBP). First irreversible reaction that is unique to glycolysis. – The commitment step - often an important control point in a pathway. It is a rate determining reaction for glycolysis pathway ATP allosterically inhibits the phosphofructokinase. – A high level of ATP in the cell means the cell does not need any more, so glycolysis is slowed down. Citric acid (intermediate of TCA cycle) is also an allosteric inhibitor. Why?  Phosphofructokinase Fructose-6-phosphate + ATP Fructose-1,6-bisphosphate + ADP Phosphofructokinase (PFK) converts fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F1,6P or FBP). Citric acid (intermediate of TCA cycle) is also an allosteric inhibitor of phosphosfructokinase. Why? The TCA cycle also provides intermediates for biosynthesis. As it is a cycle, the overall level of the intermediates is reflected in the level of citrate. If the cell has enough there is no need for any more citrate and so glycolysis slows down.  Pyruvate kinase Phosphoenolpyruvate + ADP Pyruvate + ATP Reaction Ten of Glycolysis: The reaction is essentially irreversible; a key control point  Pyruvate kinase Phosphoenolpyruvate + ADP Pyruvate + ATP 3 C molecule 3 C molecule Pyruvate kinase converts Phosphoenolpyruvate (PEP) to pyruvate Pyruvate kinase is allosterically activated by fructose-1,6- bisphosphate; this is an example of a feedforward control Pyruvate kinase is allosterically inhibited by ATP Generates second ATP by substrate level phosphorylation Glycolysis will produce 2x pyruvate from 1 glucose molecule Fate of pyruvate can vary…  Catabolism of other sugars Glycogen loses glucose residues in the form of glucose-1- phosphate. This is converted to glucose-6-phosphate by phosphoglucomutase. Other hexose sugars e.g. fructose, mannose, galactose, get converted into intermediates of the glycolytic pathway. – Galactose ends up as glucose-1-phosphate and then glucose- 6-phosphate – Fructose can end up as either fructose-6-phosphate or glyceraldehyde-3-phosphate. Varies by tissues and enzymes. Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Glycolysis generates pyruvate: Then what? Fate of pyruvate depends on presence or absence of oxygen. – And the type of cell If oxygen is present the environmental conditions for the cell are? aerobic If oxygen is absent the environmental conditions for the cell are? anaerobic  Glycolysis generates pyruvate: Then what? Under aerobic conditions cells use the enzyme pyruvate dehydrogenase to convert pyruvate to acetyl-CoA  Glycolysis generates pyruvate: Then what? Under aerobic conditions cells use pyruvate dehydrogenase to convert pyruvate to acetyl-CoA and generate NADH. Happens in mitochondria. Glycolysis can generate 2 molecules of pyruvate (from one glucose molecule) so can generate 2 NADH. NADH will then be oxidised via the electron transport chain in mitochondria (see lecture on ETC and ATP synthesis) to regenerate NAD+. Acetyl-CoA can enter the TCA Cycle (next lecture)  Glycolysis generates pyruvate: Then what? Under anaerobic conditions the cells carry out fermentation. Oxygen is not present and so cannot act as the final electron acceptor. Pyruvate acts as an organic electron acceptor. In muscle cells pyruvate is reduced to lactate In yeast pyruvate is reduced to ethanol The function of fermentation is to regenerate NAD+ so it can be used again in glycolysis. If NAD+ (oxidised form of the electron carrier, NADH) is not regenerated glycolysis will stop. Carried out by Lactobacillus and humans Carried out by yeast such as Saccharomyces. Exploited by humans. Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous).  Gluconeogenesis Gluco = glucose; neo = new; genesis = making Synthesis of glucose from simpler precursors such as pyruvate or lactate. Gluconeogenesis occurs in liver and provides glucose for export to other tissues when other sources of glucose are exhausted. Uses some of the enzymes of glycolysis (the reversible ones). Other enzymes used for non-reversible glycolysis reaction steps. Allows control of metabolism. Replacement enzymes for Gluconeogenesis  Replacement enzymes for Gluconeogenesis Pyruvate to phosphoenol pyruvate: Pyruvate kinase replaced by Pyruvate carboxylase (pyruvate to oxaloacetate) and phosphoenolpyruvate carboxykinase Fructose-1,6-bisphosphate to fructose-6-phosphate: Phosphofructokinase replaced by Fructose-1,6- bisphosphatase Glucose-6-phosphate to glucose: Hexokinase replaced by Glucose-6-phosphatase  Gluconeogenesis Replacements 6C * Glucose-6-phosphatase 6C 6C Fructose-1,6-bisphosphatase 6C * 3C + 3C 3C 3C 3C 3C Pyruvate carboxylase and Phosphoenolpyruvate 3C * carboxylase  In summary Glycolysis requires ‘energy’ initially, will then generate ‘energy’ Glycolysis has a net yield of 2x ATP, 2x NADH and 2x pyruvate. Many of the enzymes of glycolysis are reversible Some enzymes are not reversible: key control points. Aerobic conditions allow pyruvate to enter TCA cycle as Acetyl CoA. Anaerobic conditions mean pyruvate is fermented to lactate or ethanol to regenerate NAD+ to continue glycolysis. Gluconeogenesis uses the reversible enzymes of glycolysis and different enzymes for the irreversible/ control steps.  Abbreviations list ATP = adenosine triphosphate Acetyl-CoA = acetyl-coenzyme A ADP = adenosine diphosphate FMN = flavin mononucleotide UTP = uridine triphosphate FAD = flavin adenine dinuclueotide (oxidised form) UDP = uridine diphosphate FADH2 = flavin adenine dinuclueotide NAD+ = nicotinamide adenine (reduced form) dinucleotide (oxidised form) Pi = phosphate (inorganic) NADH = nicotinamide adenine dinucleotide (reduced form) G6P = glucose-6-phosphate NADP+ = nicotinamide adenine E = enzyme dinucleotide phosphate (oxidised form) S = substrate NADPH = nicotinamide adenine P = product dinucleotide phosphate (reduced form) ES = enzyme: substrate complex [ ] = concentration of Any questions: You can type in the chat function box during this live session (synchronous)? Or onto the Question Board in the Biochemistry Blackboard module and I will look at them later (asynchronous). MCQ quiz for Lecture 19: Glycolysis: a starting point of metabolism Answers will be given in your Seminar sessions – with further discussion. You must attempt before your seminar session. These quizzes are part of your learning for the Biochemistry module They will aid your on-going studies at the University of Westminster Q1) Which of the following processes take(s) place before glycolysis? A. Proteins hydrolysed to amino acids B. Starch or glycogen hydrolysed to monosaccharides C. Oxygen is reduced to water D. Glucose crosses mitochondrial outer membrane into the intermembrane space E. Small molecules broken down to pyruvate Q2) Glycolysis is a pathway of glucose that takes place in. Select the most appropriate words to fill in the blanks A. Respiration; prokaryotes only B. Anabolism; the cytosol C. Fermentation; both prokaryotes and eukaryotes D. Catabolism; cells when [ATP] is low E. Oxidation; eukaryotes only  Q3) How can molecules other than glucose enter the glycolytic pathway? A. They cannot, only glucose enters this pathway B. They are converted into intermediates of the glycolytic pathway and enter at the corresponding step C. They are converted by isomerases to glucose and enter at step 1 D. Other hexose sugars enter alternative parallel respiratory pathways instead E. They are phosphorylated and then enter glycolysis after the first irreversible step (hexokinase)  Q4) Which of the following statements is incorrect? A. Some steps in glycolysis consume ATP B. Glycolysis generates ATP C. The 6C sugar is broken down to two 3C compounds D. Glycolysis generates energy E. In glycolysis, most steps are reversible but three are essentially irreversible  Q5) Which of the following are mechanisms of regulating the activity of phosphofructokinase? 1) Allostery A. 1, 3 and 5 2) Inhibition B. 2 and 4 3) Positive feedback C. 4 and 5 4) Activation of the enzyme D. 2, 3 and 4 by phosphorylation E. 1 and 2 5) Activation of the enzyme by proteolytic cleavage