Biochemistry I Fall Term, 2000

Lecture 30: Glycolysis

Assigned reading in Campbell: Chapter 13
Lecture and notes by Dr. Eric Grotzinger

Take the Review Quiz on Lecture 30 concepts.

Glycolysis Intermediates: Chime images of the 10 reactions in the pathway.
Hexokinase Induced Fit: Chime images of the two conformations (at Univ. Arizona).
Protein Structure Examples: Chime images of Triose Phosphate Isomerase, Pyruvate Kinase, and Lactate Dehydrogenase.
Glycolysis Pathway Summary 11.15.00: Class handout on the 10 reactions in the pathway.

Central Pathways of Energy Production

Intracellular locations:

• Glycolysis - cytosol
• Fatty Acid Oxidation: Inner matrix of mitochondria
• Citric Acid Cycle: Inner matrix of mitochondria
• Oxidative Phosphorylation: Inner membrane of mitochondria

Free Energy Management:

Stored in the following ways:

• High energy chemical species (i.e. phosphoanhydride bonds in ATP)
• Redox Reactions
• Membrane potentials (concentration gradient and voltage difference)

Utilized in the following ways:

• Chemical synthesis reactions (e.g. protein synthesis, DNA synthesis
• Mechanical work (e.g. transport, muscle function)
• Electrical work (e.g. nerve conduction)

Chapters 13-15 of Campbell describes many (interesting) details of the enzyme mechanisms. For the pathways described in these chapters, you should know the following:

• Input substrates (and their structures) to the pathway.
• Output products (and their structures) from the pathway.
• Regulatory steps and how they are regulated.
• General enzymatic nomenclature.
• Cellular location of each pathway.

You will not be required to know the details of each mechanistic step and there will be little emphasis on detailed knowledge of the structures of the intermediates in the pathway. However, some knowledge of the structure of the intermediates will certainly help you to understand the chemistry associated with each enzymatic transformation. You should become familiar with each of the enzymatic steps in these pathways at a level illustrated below for glyceraldehyde-3-phosphate dehydrogenase. In this example NAD⁺ could be considered as either a substrate or a cofactor.
The lectures on these chapters will focus on several key steps in each pathway, e.g. there are four to be emphasized in glycolysis.

Enzyme Nomenclature:

Kinase: transfers a phosphate group from ATP (i.e. hexokinase, galactose kinase, pyruvate kinase)

Isomerase: converts one isomer to another (i.e. phosphogluco isomerase, triose phosphate isomerase)

Aldolase: catalyzes aldol condensation(i.e. aldolase, functions in reverse in glycolysis)

Dehydrogenase: removes hydrogens by oxidation. Usually require NAD⁺ or FAD as co-factors/co-substrates). In some cases results in formation of a "high-energy" phosphate bond by substrate level phosphorylation (i.e. glyceraldehyde-3-P dehydrogenase.

Mutase: group transfer enzyme. Common use is to move phosphates to different positions on sugars (i.e. phosphoglycerate mutase, glucose-1-P mutase).

Enolase: converts C=C group to alcohol. No change in oxidation state. Functions in reverse direction in glycolysis (enolase) and forward direction in fatty acid oxidation and in the citric acid cycle (fumarase).

Synthase: (also known as synthetase). Usually an enzyme that combines two things to make a new compound. (i.e. citrate synthase, succinyl CoA synthetase). In the citric acid cycle citrate synthase runs in the forward direction while succinyl CoA runs in the reverse direction.

ATPase: Hydrolyses ATP to ADP and P_i. This reaction runs in reverse in F_oF₁ ATPase to generate ATP using the free energy of the proton gradient.

General Features of Glycolysis:

• Glycolysis is found in all living organisms.
• Input: Glucose, 2 ATP, 4 ADP, 2 NAD⁺.
• Functions under aerobic or anaerobic conditions.
• Output (aerobic): 2 NADH, pyruvate, 2 ADP, 4 ATP.
• Output (anaerobic): lactate, 2 ADP, 4 ATP.
• Net energy gain under aerobic conditions: 2 ATP, 2 NADH.
• Rapid production of ATP from glucose.
• Lactose, sucrose, and glycogen enter the pathway above the regulatory step (PFK).
  This is an example of the convergence of diverse substrates onto a common pathway.
• All of the intermediates are phosphorylated.

Four Key Enzyme Reactions

    1. Hexokinase: Step 1

• Glucose transported into the cell by facillitated diffusion cannot diffuse out as G-6-P.
• Phosphophorylates other hexoses.
• Example of induced fit by glucose binding.
• Transfer of ATP phosphate to water is negligible.
  Water is excluded from the active site by the conformational change.
• Net energetics are favorable D G°' = -16.7 kJ/mol.

    2. Phosphofructokinase: Step 3

• Inhibited by ATP, citrate, and phosphoenol pyruvate
• Activated by ADP, AMP, cAMP, fructose-2,6-bisphosphate (F2,6P).
• Tetrameric allosteric enzyme with two ATP binding sites (one allosteric, other active site).
  1. T state (inactive) stabilized by ATP, Citrate, Phosphoenol pyruvate.
  2. R state (active) stabilized by ADP, AMP, cAMP, F2,6P.
• ATP is buffered in cells by the enzyme, adenylate kinase, that converts 2ADP to ATP + AMP. Therefore levels of ATP do not change much. Consequently, the higher levels of AMP and ADP during ATP demand largely regulate PFK activity.
• The enzyme catalyzing the back reaction (fructose-1,6- bisphosphatase) is inhibited by AMP and F2,6-Phosphate. The combined regulation of the forward and reverse pathways can result in a 100 fold change in the flux through this step.

  Note that after Step 3 glycolysis has used two ATPs for each glucose that enters the glycolytic pathway.

    3. Glyceraldehyde-3-phosphate Dehydrogenase: Step 6

• Oxidation of the aldehyde, producing 3-phosphoglycerate, is exergonic.
• Substrate-level phosphorylation is endergonic.
  1. The "high energy" bond formed is a mixed acid anhydride.
  2. P_i (HPO₄^2-) is the substrate.
     HAsO₄^2- (arsenate) creates a bypass of the ATP-forming Step 7 that follows.
• The energetics of this oxidation-driven phosphorylation reaction result in a net reversible reaction.

    4. Pyruvate Kinase: Step 10

• Tetrameric allosteric enzyme; inhibited by ATP.
• ATP produced in this last step represents the net yield from anaerobic hydrolysis.

The Fate of Pyruvate

• Anaerobic tissues and/or organisms must recycle NADH to NAD⁺.
  1. Muscles: pyruvate is reduced to lactate; or
  2. Yeast: pyruvate is decarboxylated to acetaldehyde and then reduced to ethanol.
  3. Fermentations in other microorganisms produce a variety of valuble commercial chemicals. These are all "waste products" to the organisms that produce them.
• Aerobic tissues and/or organisms capture the energy in NADH.
  Pyruvate is decarboxylated and converted to acetyl Coenzyme A before entering the TCA cycle (Chapter 15).

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