Glycolysis Two stage process Stage 1 – trapping and destabilising glucose in order to produce 2x3c molecules (5steps in the process). Requires energy (2 ATPs) Stage 2 – oxidation of the 3c molecules to pyruvate (5steps in the process). Energy generated (4tps and 2 NADH) Stage 1 Step 1 – trapping glucose, glucose enters via facilitated diffusion through specific transport proteins. The family of transporters is known as GLUT, GLUT 3 (brain, nerve tissue) Low Km allows relatively constant rate of glucose uptake, GLUT 2 (liver, ? -cells pancreas) high Km rate of uptake proportional to extracellular glucose concentration.
GLUT4 (Muscle and adipocytes) rate of uptake controlled by insulin. Does not alter Km of the protein, but changes Vmax by increasing the number of transporters,. Number of GLUT4 transporters can be increased by exercise. Transporters are found in intracellular pods, when glucose levels rise, insulin is activated which creates a cascade of reactions which brings the pool of transporters to the surface leading to more uptake of glucose. Once in the cell glucose is trapped by phosphorylation. Glucose 6-phosphate is negatively charged and cannot diffuse out of the cell.
Addition of the phosphate group begins the destabilisation process of glucose, which leads to further metabolism hexokinase Can phosphorylate (kinase) a variety of hexose (six carbon) sugars (glucose, mannose even fructose) Induced fit enzyme action Equilibrium of reaction strongly favours glucose 6-phospate (effectively irreversible reaction) Regulatory enzyme of glycolysis, inhibited by glucose 6-P (FEEDBACK INHIBITION) Step 2 – formation of fructose 6-phosphate Isomerisation of Glucose 6-P to Fructose 6-P is a completely reversible reaction carried out by the enzyme phosphoglucose isomerase.
Convert from one isomer (glucose) to another (fructose) by Tautomerisation Step 3 – second phosporylation reaction The enzyme Phosphofructokinase carries out this reaction. Inhibited by ATP, Citrate and H+ ions Stimulated by AMP, ADP and Fruc 2,6-bisP Steps 4 and 5 – Splitting Fructose 1,6-bisP into useful 3C fragments. Cleavage of Fructose 1,6-bisP is catalysed by the enzyme Aldolase to yield 2 Triose phosphates Readily reversible under normal physiological conditions Glyceraldehyde 3-P is on the direct pathway of glycolysis. DHAP is not.
DHAP needs to be converted into G 3-P otherwise a 3C fragment capable of generating ATP will be lost. The enzyme Triose Phosphate isomerase (TIM) catalyses this reversible reaction. At equilibrium 96% is in the DHAP form. However because of subsequent reaction of glycolysis and removal of Glyceraldehyde 3-P the equilibrium is pushed towards its formation. Triose Phosphate Isomerase (TIM Great catalytic prowess, accelerates isomerisation by a factor of 1010 compared to simple base catalysis Kinetically perfect enzyme, the rate limiting step is the diffusion-controlled encounter of substrate and enzyme.
So 2 molecules of G-3-P almost simultaneously from F 1,6-bisP Stage 2 Step 6 – formation of a high energy bond G 3-P is oxidised and phosphorylated by the enzyme G 3-P dehydrogenase. Dehydrogenase transfer “high energy” electrons from complex organic molecule to NAD+ to form NADH The resulting intermediate 1,3-bisphosphosphoglycerate is an acyl phosphate i. e. has a high-phosphoryl-transfer potential. Sum of two processes Oxidation of the aldehyde to a carboxylic acid by NAD+ Joining of orthophosphate to the carboxylic acid Step 7: ATP generation from 1,3-bisPglycerate
Substrate level Phosphorylation Remember Glucose (6C) yields 2 x 3C intermediates therefore 2 ATP’s generated per glucose molecule. Steps 8, 9 and 10: Generation of additional ATP and pyruvate formation (2 per glucose molecule) Phosphoryl group on 3-Pglycerate shifts position, followed by dehydration and formation of a C=C bond. Increases transfer potential of phosphoryl group. Pyruvate kinase Irreversible transfer of phosphoryl group to form ATP Substrate level phosphorylation Regulatory enzyme activated by Fructose 1,6bisP and inhibited by ATP and alanine.
Glycolysis would not proceed for long if it the pyruvate was final metabolite as the redox balance of the cell would not be maintained Limited amounts of NAD+ in cells. Must REPLACE!! Fates of pyruvate During glycolysis NAD+ is converted to NADH Glycolysis cannot continue if [NAD+] decreases. Oxygen present – electrons on NADH transferred to oxygen (via electron transport chain) to produce H2O, ATP and NAD+. No oxygen present – Electrons on NADH transferred to pyruvate (or intermediate) to form lactate or ethanol and NAD+(recycled for step 6 of glycolysis)
Ethanol Formation: Yeast and some other microorganisms Anaerobic process (no O2 required) Glucose + 2 H+ + 2 ADP + 2 Pi 2 Ethanol + 2 CO2 + 2 ATP + 2 H2O Organisms when oxygen is limited e. g. intense exercise Regeneration of NAD+ for step 6 of glycolysis Under aerobic conditions much more energy can be extracted by means of the TCA cycle and OX PHOS Pyruvate enters mitochondria and is oxidised to acetyl CoA. NADH generated at step 6 of glycolysis cannot enter the mitochondria, so NAD+ is regenerated indirectly by OX PHOS using specific shuttles.