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14.1: Glycolysis

  • Page ID
    15004
  • Glycolysis: Anerobic Coupling of Oxidation and ATP Synthesis

    Our main goal is to understand how oxidation reactions can lead to ATP synthesis. First let us consider ATP production under anaerobic condition, such as which often occurs during the fight or flight response. You know how terribly you feel when you run a 100 m dash. Your muscles ache due to lactic acid buildup, and you know you can't seem to get enough dioxygen into your body. Under these conditions, a pathway called glycolysis (which you studied in biology) is active. In this pathway, glucose, a 6 carbon hexose, is converted to two, 3C molecules - pyruvate. The figure below shows the entire pathway using Lewis wedge/dash representations.  For select this link for a more traditional representation using Fischer projections

    Patty:  I updated most cdx files on this page on 8/23/21 using SVG files from cdx files. They stay sharp even on zoom. 

    metabolismWP_Glycolysis0821.svg.

     

    Now lets examine each step in more detail. Add mechanisms later.

    Reaction 1: Glc → Glc6P. ΔGo=-4.0 kcal/mol.

    Glycolysis_Rx1.svg

    Function: By placing a phosphate with its charges on glucose, the molecule is less likely to leave cell. All subsequent products within glycolysis are phosphorylated. This reaction is a nucleophilic substitution reaction on the gamma phosphate of ATP. A phosphoanhydride bond is broken in ATP as a phosphoester bond is made in glucose 6-P. Hence the reaction proceeds with a negative ΔGo.

    Reaction 2: G6P ↔ F6P. ΔGo=+0.4 kcal/mol

    We'll present this reaction in two forms, wedge dash and Fischer projections. 

    Glycolysis_Rx2B.svg

     

     

     

     

     

    Glycolysis_Rx2Fischer.svg

    Function: From the linear Fischer structure, it is evident that the C=O has been moved to C2. The carbonyl O is now position to be an electron sink facilitating electron flow for reaction 4. This isomerization reaction would be expected to have a ΔGo of about 0.

    Reaction 3: F6P → F 1,6 BP. ΔGo=-3.4 kcal/mol

    Glycolysis_Rx3.svg

    Function: By phosphorylating this intermediate, both products of the cleavage of this 6C molecule will be phosphorylated, keeping both more readily inside the cell. This reaction is a nucleophilic substitution reaction on the gamma phosphate of ATP. A phosphoanhydride bond is broken in ATP as a phosphoester bond is made in F1,6BP. As in reaction 1, this reaction proceeds with a negative ΔGo

    Reaction 4: F 1,6 BP ← DHAP + G3P. ΔGo=-3.4 kcal/mol = + 5.7 kcal/mol

    We'll present this reaction in two forms, wedge dash and Fischer projections

    Glycolysis_Rx4.svg

     

    Glycolysis_Rx4Fischer.svg

    Function: This is the first C-C bond cleavage within glucose on the path to cleave of all the rest in subsequent pathways. This reaction is the reverse of an aldol condensation when an enol or enolate reaction with a carbonyl C to form an adduct. Note that both products are phosphorylated. The reaction is not thermodynamically favored but is pull in the forward direction by subsequent reactions in the pathway.

    Reaction 5: DHAP ↔ G3P. ΔGo= +1.8 kcal/mol

    Glycolysis_Rx5.svg

    Function: This is another simple isomerization reaction. Only one product, glyceraldehyde 3P continues on in glycolysis, so only one enzyme is needed to metabolize the cleavage products of this reaction further. As in other isomerization reactions, the ΔGo is close to 0.

    Reaction 6: G3P ↔1,3 BPG. ΔGo= +1.5 kcal/mol

    Glycolysis_Rx6.svg

     

    Function: This is a big reaction! Note that the ΔGo is close to 0 but look a what really happened. The carbonyl O in G3P has been oxidized to the form of a mixed anhydride which can donate a phosphate to ADP (in the next step) to form ATP. That this is an oxidation reaction should be obvious from the fact the the carbonyl C in G3P has two bonds to O but 3 bonds in 1,3 BPG.

    To carry out an oxidation reaction, you need an oxidizing agent. In comes NAD+, a modest put very prevalent oxidizing agent in biology. When glucose is oxidized completely by O2 to CO2 during combustion, much energy is released so we can surmise that oxidation reactions, if carried out by powerful oxidizing agents like O2, proceed with a large negative ΔGo. For every oxidation reaction, the oxidizing agent is reduced. All reactions are potentially reversible so the products formed are new potential oxidizing and reducing agents. As in acid/base reactions, which proceed from stronger acid to weaker conjugate acid, redox reactions proceed from stronger to weaker oxidizing agent. For reaction 6, we must use tables of redox potentials to calculate the actual ΔGo. It turns out to be close to 0 which is great since in the same reaction, a substrate-level phosphorylation reaction (using inorganic phosphate - Pi instead of ATP) occurs. In summary this reaction catalyzes the first and only oxidation of glucose in glycolysis which has paid (thermodynamically) for the generation of a mixed anhydride whose phosphorylating potential is higher that that of ATP.

    Reaction 7: 1,3 BPG + ADP + H+ → 3PG + ATP ΔGo= -4.5 kcal/mol

    Glycolysis_Rx7.svg

    Function: It's finally happened. An ATP has been made for each of the two 1,3-BPG molecules derived from Glc. We've made back the ATP used in steps 1 and 3. A mixed phosphoanhydride bond is broken in 1,3 BPG as a phosphoanhydride bond is made in ATP. As the mixed phosphoanhydride has higher energy than its hydrolysis product compared to the phosphoanhydride in ATP, the reaction proceeds with a negative ΔGo

    Reaction 8: 3PG ↔ 2PG ΔGo= +1.1 kcal/mol

    Glycolysis_Rx8.svg

     

    Function: This isomerization reaction proceeds with little thermodynamic barrier. It's function is to locate the phosphate on C2 which on the next reaction (dehydration) will form a molecule whose phosphoryl transfer potential is greater than ATP.

    Reaction 9: 2PG ↔ PEP + H2O ΔGo= +0.4 kcal/mol

    Glycolysis_Rx9.svg

     

    Function: Now you can see the rationale for reaction 8. By a simple and thermodynamically simple dehydration, a molecule with high phosphoryl transfer potential (see dotted red box) has been produced which in the next and final step of glycolysis produces ATP.

    Reaction 10: PEP + ADP → Pyr + ATP ΔGo= -7.5 kcal/mol

    Glycolysis_Rx10.svg

     

    Function: In this step, 1 more ATP is made for each PEP consume (hence 2 ATPs for both 3C PEPs). The phosphoryl transfer potential for PEP is higher than than for ATP, which allows this reaction to proceed with a large negative ΔGo (-7.5 kcal/mol).

    This is the net reaction of the glycolytic pathway:

    Glc + Pi + 2ADP + 2NAD+ → 2 Pyr + 2 ATP + 2NADH + 2H+ + 2H2O

    Since we most interested in energy transduction at this point, let's consider just two important step in glycolysis that directly lead to ATP synthesis. Only one oxidative step is found in this pathway, namely the oxidative phosphorylation of the 3C glycolytic intermediate glyceraldehyde-3-phosphate, to 1,3-bisphosphglyercate, a mixed anhydride (see link below for mechanism). The oxidizing agent is NAD+ and the phosphorylating agent is NOT ATP but rather Pi. The enzyme is named glyceraldehyde-3-phosphate dehydrogenase. It contains an active site Cys, which helps explain how the enzyme can be inactivated with a stoichiometric amounts of iodoacetamide. A general base in the enzyme abstracts an H+ from Cys, which attacks the carbonyl C of the glyceraldehyde, forming a tetrahedral intermediate. Instead of the expected reaction (which would be the protonation of the alkoxide in an overall nucleophilic addition reaction at the aldehyde), a hydride leaves from the former carbonyl C to NAD+ in an oxidation step. Notice, this is a two electron oxidation reaction similar to seen in alcohol dehydrogenase. An acyl-thioester intermediate has formed, much like the acyl intermediate that formed in Ser proteases. Next inorganic phosphorous, Pi, attacks the carbonyl C of the intermediate in a nucleophilic substitution reaction to form the mixed anhydride product, 1,3-bisphoshphoglycerate. Although we have formed a mixed anhydride, we cleaved a sulfur ester, which is destabilized with respect to its hydrolysis products (since the reactant, the thioester, is not stabilized by resonance to the extent of regular esters owing to the poor donation of electrons from the larger S to the carbonyl-like C.) In the next step, catalyzed by the enzyme phosphoglycerate kinase, ADP acts an a nucleophile which attacks the mixed anhydride of the 1,3-bisphosphoglyerate to form ATP. Note that the enzyme is name for the reverse reaction. We have coupled oxidation of an organic molecule (glyceraldehyde-3-phosphate) to phosphorylation of ADP through the formation of a "high" energy mixed anhydride, 1,3-bisphosphoglycerate.

    The linkage between oxidation of glyceraldehyde-3-phosphate and the phosphorylation of ADP by 1,3-bisphosphoglycerate can be artificially uncoupled by adding arsenate, which has a similar structure as phosphate. The arsenate can form a mixed anhydride at C1 of glyceraldehyde-3-phosphate, but since the bringing O-As bond is longer and not as strong as in the mixed anhydride, it is easily hydrolyzed. This prevents subsequent transfer of phosphate to ADP to form ATP.

    Figure: MECHANISM OF OX-PHOS IN GLYCOLYSIS: GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND 3-PHOSPHOGLYCERATE KINASE

    update to wedge/dash

    oxphosglycol.gif

    iconChime.gifJmol: Updated Glyceraldehyde-3-phosphate dehydrogenase (NAD) Jmol14 (Java) | JSMol (HTML5)

    Summary: Under anaerobic conditions, glucose (6Cs) is metabolized through glycolysis which converts it to two molecules of pyruvate (3Cs). Only one oxidation step has been performed when glyceraldehyde 3-phospate is oxidized to 1,3-bisphosphoglycerate. To regenerate NAD+ so glycolysis can continue, pyruvate is reduced to lactate, catalyzed by lactate dehydrogenase. These reactions take place in the cytoplasm of cells actively engaged in anaerobic oxidation of glucose (muscle cells for examples during sprints). Note that the enzyme is named for the reverse reaction, the oxidation of lactate by NAD+.

    Glycolysis_LactDehydro.svg

     

    Lactate in the muscle can go by way of the blood to the liver (where NAD+ is not depleted) and be converted back to pyruvate and eventually back to glucose through a pathway called gluconeogenesis. The liver can export the glucose into the blood from where it can be taken up by the muscle for ATP production. This cycle is called the Cori cycle.

    Coricycle.svg

     

    Enzyme

    Wikipedia Link

    EC #

    EC-PDB

    KEGG

    MAP

    BRENDA

     

    PDB-E

    PDB-E
    SUMM

    RCSB-PDB

    Hexokinase

    2.7.1.1

    Hexokinase

    KEGG

    BRENDA

    2.7.1.1

    1HKB

    1HKB

    Phosphoglucose Isomerase

    5.3.1.9

    Phosphoglucose Isomerase

    KEGG

    BRENDA

    5.3.1.9

    1IAT

    1IAT

    Phosphofructokinase

    2.7.1.11

    Phosphofructokinase

    KEGG

    BRENDA

    2.7.1.11

    -

     

    Aldolase

    4.1.2.13

    Aldolase

    KEGG

    BRENDA

    4.1.2.13

    1ALD

    1ALD

    Triose Phosphate Isomerase

    5.3.1.1

    Triose Phosphate Isomerase

    KEGG

    BRENDA

    5.3.1.1

    1HTI

    1HTI

    Glyceraldehyde-3P dehydrogenase

    1.2.1.12

    Glyceraldehyde-3P dehydrogenase

    KEGG

    BRENDA

    1.2.1.12

    1U8F

    1U8F

    Phosphoglyceratekinase

    2.7.2.3

    Phosphoglyceratekinase

    KEGG

    BRENDA

    2.7.2.3

    2WZB

    2WZB

    Phosphoglyceratemutase

    5.4.2.1

    Phosphoglyceratemutase

    KEGG

    BRENDA

    5.4.2.1

    1LJD

    1LJD

    Enolase

    4.2.1.11

    Enolase

    KEGG

    BRENDA

    4.2.1.11

    1TE6

    1TE6

    Pyruvate kinase

    2.7.1.40

    Pyruvate kinase

    KEGG

    BRENDA

    2.7.1.40

    1LIU

    1LIU

    Lactate dehydrogenase

    1.1.1.27

    Lactate dehydrogenase

    KEGG

    BRENDA

    1.1.1.27

    1I0Z

    1I0Z

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