Glucose Metabolism Glycolysis Biochem 4511 Figures Essential Biochemistry 3rd Ed Pratt and Cornely Principles of Biochemistry 5th Ed Moran et al Glucose Metabolism Glycolysis the breakdown of glucose to pyruvate to yield energy Gluconeogenesis the combination of smaller carbon containing compounds to synthesize glucose Glycogen synthesis and breakdown storage or recovery of glucose from glycogen polymer Glycolysis Overview Glycolysis occurs in a total of 10 steps Glucose 6 carbon molecule is broken down into two pyruvates 3 carbon molecules The first 5 steps are the energy investment phase 2 ATP molecules are hydrolyzed to ADP per glucose The second 5 steps 6 10 are the energy payoff phase 4 ATP molecules are generated per glucose for a net gain of 2 ATP 2 NAD electron carriers are reduced to 2 NADH NADH can subsequently be oxidized to generate additional ATP Electron Transport Chain Energy Investment Phase Energy Payoff Phase Step 1 Hexokinase Reaction Kinases are enzymes that phosphorylate molecules ATP is invested ATP hydrolysis drives the reaction Reaction is irreversible Glucose 6 phosphate is an intermediate for several metabolic pathways NOT committed to glycolysis Step 2 Phosphoglucose Isomerase Reaction Conversion of an aldose glucose to a ketose fructose Conversion from a pyranose to a furanose ring This reaction is near equilibrium and reversible Still NOT committed to glycolysis Step 3 Phosphofructokinase Reaction A second ATP is invested and ATP hydrolysis drives the reaction This reaction is irreversible This is the first committed step of glycolysis and it is HIGHLY regulated next lecture Fructose Exists in Both Ring and Linear Forms Within the Cell In the next step of glycolysis fructose 1 6 bisphosphate will be cleaved into two three carbon sugars It is easier to visualize this reaction using the linear form of the molecule Step 4 Aldolase Reaction The aldolase reaction is the reverse of an aldol condensation One glucose 6 carbon molecule is broken down into 2 three carbon sugars Aldolase Mechanism in Detail 1 Nucleophilic attack of carbonyl by the lysine amine yields a covalent intermediate 2 Subsequent loss of water completes the formation of a Schiff Base covalent catalysis 3 Tyrosine abstracts proton from alcohol base catalysis Aldolase Mechanism in Detail 3 Cleavage of C3 C4 bond releases glyceraldehyde 3 phosphate GAP 4 Tyrosine protonates C3 reforming the Schiff base acid catalysis 5 Hydrolysis of Schiff base regenerates the active site and releases dihydroxyacetone phosphate DHAP Step 5 Triose Phosphate Isomerase Reaction Reversible interconversion of dihydroxyacetone phosphate DHAP to glyceraldehyde 3 phosphate GAP Two GAP molecules proceed through remainder of glycolysis G 0 for the conversion of DHAP to GAP but this reaction proceeds forward because GAP is quickly consumed in the next step of glycolysis Why is it desirable to convert DHAP to GAP Step 5 Triose Phosphate Isomerase Reaction Even though G 0 this reaction proceeds forward because GAP is quickly consumed in the next step of glycolysis At any given time 94 of the molecules are DHAP and 6 are GAP but any GAP generated is quickly converted to 1 3 BPG in the next step of glycolysis Triose phosphate isomerase is considered a perfect enzyme as its rate of catalysis is diffusion limited Significant transition state stabilization provided by conformational changes of the enzyme upon binding green Step 6 GAP Dehydrogenase Reaction Importantly the phosphate does NOT come from ATP and proceeds through a high energy thioester intermediate NAD is reduced to NADH Reaction is both a phosphorylation and an oxidation reduction reaction Reaction is strongly inhibited by AsO4 3 which competes with PO4 2 for binding the enzyme GAP Dehydrogenase Mechanism Step 7 Phosphoglycerate Kinase Reaction ATP is generated through substrate level phosphorylation Since there are two 1 3 BPG molecules per glucose this reaction occurs twice and the initial 2 ATP molecules have been recouped from the energetic investment At this step the net yield of ATP through glycolysis is ZERO Step 8 Phosphoglycerate Mutase Reaction Phosphate group is moved from C 3 to C 2 Moving the phosphate closer to the carboxylic acid increases the energy of the phosphoanhydride bond Isomerization of 3 phospho glycerate is catalyzed by a phosphorylated histidine at the active site Carboxylic acid is stabilized by adjacent lysine side chain Step 9 Enolase Reaction Enolase catalyzes a dehydration reaction to yield the enol form of the substrate The generated phosphorylated enol is a high energy species with enough energy to transfer the phosphate to ADP in the next step Step 10 Pyruvate Kinase Reaction ATP formed by a second substrate level phosphorylation The final energetic payoff nets 2 ATP per glucose for the full glycolysis cycle The initial product is the enol form of pyruvate which tautomerizes to pyruvate Glucose six carbons has been broken down into two pyruvates 2x three carbon molecules Free Energy Changes of Glycolysis Go 0 G 0 Based on Standard Gibbs free energy changes several steps of glycolysis are disfavored Cellular concentrations of metabolites ensure that all reactions are spontaneous under physiological conditions Free Energy Changes of Glycolysis Steps 1 3 and 10 are irreversible forward only reactions Step 5 is endergonic but driven forward by the GAP concentration gradient Regulation of Glycolysis Biochem 4511 Figures Essential Biochemistry 3rd Ed Pratt and Cornely Principles of Biochemistry 5th Ed Moran et al Lehninger Principles of Biochemistry 5th Ed Nelson Cox Biochemistry 3rd Ed Voet Voet Fundamentals of Biochemistry 2nd Ed Voet Voet Pratt Regulation of Metabolic Pathways Product Inhibition The final product in a metabolic pathway commonly inhibits an enzyme allosterically earlier in the pathway to prevent product accumulation Logically if there are already sufficient quantities of the final product of a pathway there is no need to invest energy and cellular resources to generate additional product Regulation of Metabolic Pathways Feed Forward Activation An early product in a metabolic pathway after the committed step commonly activates an enzyme allosterically later in the pathway to increase final product formation Logically if there is a backlog of an intermediate product there is a need to invest energy and cellular resources to increase the rate of the overall pathway Regulation of Metabolic Pathways Covalent