Cellular respiration 2

Cellular respiration 2

1. Learning objectives

   • Overview of the oxidation of glucose by glycolysis to produce pyruvate
   • Control of glycolysis to meet the energy demands of the cell
   • The role of creatine phosphate in short term energy generation
   • The fate of pyruvate in aerobic and anaerobic conditions: the generation of acetyl-CoA and lactate
   • Control of pyruvate dehydrogenase and its importance

2. Glucose as a fuel

   • Glucose = important fuel molecule in most cells
   • Cellular glucose - transported into cells via glucose transporters (GLUT)
     • GLUT transporter proteins are transmembrane proteins which catalyse the facilitative diffusion of glucose down its concentration gradient
       • Concentration gradient maintained by rapid cellular metabolism of glucose
     • There are 4 GLUT isoform - varying affinities for glucose and different tissue distributions

3. GLUT transporter structure

   • Highly hydrophobic side chains, polypeptide clustering
   • Some groups glycosylated = addition of sugar
   • Glucose transport into the cytoplasm
     • Conformational change allows glucose to be released into cytoplasm
       • Conformational change = change of shape of macromolecule, often induced by environmental factors. - macromolecules are often flexible and dynamic.

4. The GLUT family members

   • GLUT1 - most cells, inc. RBCs. Intermediate Km.
     • Ubiquitous = found everywhere
   • GLUT2 - liver and pancreatic B-cells. High Km
   • GLUT3 - mainly brain. Low Km.
   • GLUT4- Primarily muscle and adipose - intermediate Km. INSULIN SENSITIVE
     • Found intracellularly in presence of insulin
   • Low Km = high enzyme affinity = less substrate required for enzyme to be active at maximum rate. High Km = requires more substrate for enzyme to be maximumly active.
   • GLUT5- fructose transporter- primarily found in intestine and kidney

5. Overview of glycolysis

   • Glucose is oxidised through the pathway of glycolysis to form 2 pyruvate molecules
     • glucose (C6H12O6) + 2NAD+ + 2ADP +Pi --> 2pyruvate (C3H4O3) + 2NADH + 2H+ +2ATP +2H20
     • small net ATP gain for each molecule of glucose oxidised
   • Cytostolic pathway occuring in all cells
     • cytostolic = occurs in the cytoplasm
   • Unusually, it can occur aerobically and anaerobically
   • 10 enzyme catalysed steps which can be split into two phases - PREPARATORY phase and PAYOFF phase

6. Control of glycolysis

   • Glycolysis has 3 key rate limiting steps - control flux through the pathway
   • HEXOKINASE, PHOSPHOFRUCTOKINASE, PYRUVATE KINASE
     • control the 3 R.L steps - allow rate of glycolysis determined by ATP demand of cells
     • Catalyse exorgenic reactions which are essentially irreversible
     • Reactions involved have -ve G.

7. Control 1: Hexokinase

   • glucose + ATP --> glucose 6P + ADP
   • HK relatively high affinity for glucose, inhibited by the reaction product G-6-P
     • acts as a non-competetive inhibitor
     • Allosteric regulation, -ve feedback inhibition
   • HK gene expression subject to regulation by hormones
   • LIVER and PANCREATIC B CELLS contain isoenzyme glucokinase GK, lower glucose affinity for glucose than HK.
     • GK regulated differently, not subject to product inhibition
   • GK only catalyses glucose phophorylation when [glucose]blood is high

8. Control 2: Phosphofructokinase-1

   • Fructose-6-P + ATP --> fructose-1,6-BP +ADP
   • PFK-1 subject to complex regulation, allows glycolysis to respond to energy status
   • Glycolysis slows when ATP demand drops and ATP high in cell. vice versa
   • Fructose 2,6-BP important activator of PFK-1 (allosteric activator)
   • Citrate and H+ inhibit enzyme - build up during anaerobic respiration
   • PFK-1 gene expression regulated by INSULIN (activated) and GLUCAGON (inhibits) - long term regulation, opposing effects

9. Summary of glycolysis regulation

   • glc-6-p allosterically inhibits hexokinase
     • Insulin activates pfk-2 while glucagon inhibits pfk-2
       • F-2,6-BP and AMP,ADP activate PFK1, while citrate, ATP and H+ inhibit PFK-1
         • F-1,6-BP activates PK, while ATP and alanine inhibit PK

10. Control 3: Pyruvate kinase

    • PEP + ADP --> pyruvate + ATP
    • PK catalyses final reaction in glycolysis and produces ATP by substrate level phophorylation
    • PK allosterically inhibited by ATP and alanine
    • PK allosterically activated by F-1,6-P2 - feed forward activation, ensures intermediates of glycolysis do not accumulate.
    • PK regulated by phosphorylation in response to hormones
      • GLUCAGON PHOSPHORYLATES - inhibit
      • INSULIN DEPHOSPHORYLATES - activate
    • PK deficiencies in RBCs cause haemolytic anaemia
      • see practicals

11. Pathways that link with glycolysis

    • Glycogen --> glucose 1-P --> glucose 6-P
    • Glucose 6P --> pentose phosphate pathway (PPP)
      • produces large amount of cofactor NADPH as well as other sugars
    • 1,3-BPG --> 2,3-BPG
      • 2,3-BPG regulates O2 binding affinty of haemoglobin, Hb

12. Aerobic v anaerobic glycolysis

    • Anaerobic - pyruvate converted to LACTATE to generate NAD+ so glycolysis can continue
      • using LACTATE DEHYDROGENASE enzyme
      • lactate metabolised by liver
      • NADH builds up if not oxidised by mitochondria in oxidative phosphorylation
    • Aerobic - pyruvate converted to acetyl-coA using PYRUVATE DEHYDROGENASE

13. The importance of anaerobic glycolysis

    • Anerobic glycolysis important in:
      • RBCs - do not contain mitochondria
        • RBCs are entirely reliant on glycolysis
      • Anaerobically respiring muscle - intense exercise
      • Tumour cells - O2 low in centre
      • Hypoxic conditions
    • Generates 2ATP per glucose, lactate must be removed to prevent ACIDOSIS
    • Lactate transported out of cell and metabolised by LIVER

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