Glycolysis (Continued), Energetics, Beer's Law, and Introduction to Glycogen

Lecture Overview

This lecture completes the glycolysis pathway from the aldolase reaction through lactate dehydrogenase, then covers alcoholic fermentation in yeast. The lecturer introduces NAD structure and its spectral properties, allosteric regulation of glycolytic enzymes, and the dual purpose of glycolysis (ATP production and biosynthetic intermediates). The middle portion addresses thermodynamic concepts (equilibrium constant, delta G, delta G prime) and Beer's Law as applied to enzyme assays, including an IU calculation for lactate dehydrogenase. The lecture concludes by transitioning to starch and glycogen structure and the early steps of glycogen biosynthesis via glycogenin and glycogen synthase.

Key Concepts and Definitions

• Aldol condensation: The chemical reaction at the active site of aldolase; in the reverse direction, it forms fructose 1,6-bisphosphate from DAP and G3P.
• Triosephosphate isomerase (TPI): Enzyme that converts DAP into G3P; catalytically perfect, limited only by diffusion.
• Catalytically perfect enzyme: An enzyme that cannot evolve to go any faster; the only rate limitation is substrate diffusion.
• Dehydrogenase: An enzyme that transfers electrons onto NAD, FMN, or FAD.
• Kinase: An enzyme that transfers phosphate groups.
• 1,3-Bisphosphoglycerate (1,3-BPG): A high-energy acyl phosphate intermediate of glycolysis; delta G prime of approximately minus 12 kcal/mol.
• Delta G prime: Free energy change when all species are at 1 molar concentration except protons, which are at pH 7.
• Mutase: An enzyme that moves a group from one position on a molecule to a different position.
• PEP (phosphoenolpyruvate): A very high-energy phosphorylated compound; delta G prime of minus 13.8 kcal/mol; its instability is due to the phosphate group preventing keto-enol tautomerization.
• Coenzyme: A small organic molecule that must be present at the enzyme active site for function.
• TPP (thiamine pyrophosphate): A coenzyme required by pyruvate decarboxylase.
• Allosteric enzyme: An enzyme that gives an S-shaped (sigmoidal) curve when velocity is plotted versus substrate; typically has multiple subunits and catalyzes reactions with large negative delta G values.
• K-equilibrium prime: The equilibrium constant adjusted for pH 7 conditions.
• Delta G naught: Free energy change when all species are at 1 molar; equals minus RT times the natural log of K-equilibrium.
• Beer's Law: A = epsilon x L x C; absorbance equals molar extinction coefficient times path length times concentration.
• Molar extinction coefficient (epsilon): A constant determined by the structure of the absorbing molecule; must be a large number; units are M^-1 cm^-1.
• Specific absorbance (little a): Used in place of epsilon when the molecular weight of the absorbing species is unknown.
• International unit (IU): Micromoles of product formed (or substrate consumed) per minute.
• Glycosidic bond: A covalent bond between carbohydrates.
• Alpha-1,4 linkage: Bond between carbon 1 and carbon 4 of glucose residues, with the bond below the plane of the ring.
• Alpha-1,6 linkage: Branch-point bond between carbon 1 and carbon 6 of glucose residues, with the bond below the plane of the donating glucose.
• Reducing end: The terminal glucose in a polymer whose anomeric bond can open and close (hemiacetal); can reduce metal ions.
• Non-reducing end (NRE): A terminal glucose whose anomeric bond cannot open and close (acetal); protected from oxidation.
• Glycogenin: A dimeric protein that initiates glycogen synthesis; attaches glucose to a tyrosine hydroxyl group and builds an 8-mer primer using UDP-glucose.
• Synthase vs. synthetase: A synthetase uses a nucleoside triphosphate (e.g., ATP); a synthase does not.
• Glycogen synthase: Enzyme that extends glycogen chains in alpha-1,4 linkages using UDP-glucose; requires the glycogenin-built primer to function.

Chronological Lecture Notes

Reference figure · external source

1. Review: Aldolase and the Aldol Condensation

• Aldolase cuts fructose 1,6-bisphosphate between carbons 3 and 4; the reaction is reversible.
• In the reverse direction, aldolase positions DAP next to G3P and catalyzes an aldol condensation to form fructose 1,6-bisphosphate.
• The lecturer noted that cells have been performing aldol condensations for billions of years.
• In the forward (breakdown) direction, one molecule of glucose yields one molecule of DAP and one molecule of glyceraldehyde-3-phosphate (G3P), each phosphorylated and each three carbons.

2. Triosephosphate Isomerase (TPI)

• TPI instantly converts DAP into G3P, so glucose effectively produces two molecules of G3P.
• TPI is one of the rare catalytically perfect enzymes: it cannot evolve to go faster, and the only rate limitation is diffusion of substrate into and product out of the active site.
• The importance of TPI: without it, half the carbons of glucose would be lost.

3. G3PDH and Formation of 1,3-BPG

• G3P + inorganic phosphate (Pi) + NAD --[G3PDH]--> NADH + H+ + 1,3-bisphosphoglycerate (1,3-BPG)
• Three substrates, three products; because TPI produces two G3P molecules, this reaction occurs twice per glucose.
• Dehydrogenases transfer electrons onto NAD, FMN, or FAD. Kinases transfer phosphate groups.
• When NAD is written close to the equilibrium arrow, the products (NADH + H+) appear above.
• 1,3-BPG is the cousin of 2,3-BPG, which is important for hemoglobin function.
• 1,3-BPG contains a high-energy acyl phosphate bond. Delta G prime is approximately minus 12 kcal/mol.
• If water were used instead of inorganic phosphate, the oxidation of the aldehyde group to a carboxylic acid would lose much of the energy as heat and entropy. By using Pi, cells trap the energy in the acyl phosphate, enabling ATP synthesis in the next step.
• The proton released comes from inorganic phosphate (which has an OH group at physiological pH).
• NAD is reduced by a hydride (H minus): the hydrogen and its two bonding electrons from the substrate carbon are transferred to NAD.

Reference figure · external source

4. Delta G Prime Notation

• Delta G prime means all species are at 1 molar except protons, which are at pH 7.
• At 1 molar proton concentration, pH would be zero and the enzyme would not function.
• The delta G prime for ATP synthesis is positive 7.7 kcal/mol.

Exam emphasis: The lecturer stated that numerical delta G values given in lecture are for reference; on exams, these values will be provided if needed.

5. Phosphoglycerate Kinase and Net ATP Balance

• 1,3-BPG + ADP --[Phosphoglycerate kinase]--> 3-PGA + ATP
• Unlike hexokinase and PFK1, phosphoglycerate kinase catalyzes a totally reversible reaction; directionality depends on actual concentrations in the cell.
• 3-PGA: the carboxyl group is carbon 1, the phosphate is on carbon 3.
• Student interaction: When asked about net ATP at this point starting from one glucose, a student answered one. The lecturer corrected: two ATPs were consumed earlier (hexokinase, PFK1), and two are produced here (one per G3P, times two G3Ps), so the net is zero. Glycolysis has broken even at this stage.

6. Phosphoglycerate Mutase

• 3-PGA --[Phosphoglycerate mutase]--> 2-PGA
• 3-PGA and 2-PGA are structural isomers.
• The mutase moves the phosphate from carbon 3 to carbon 2 using a shell game mechanism: the active site already contains a phosphate before binding substrate. The phosphate placed on carbon 2 is not the same phosphate that was on carbon 3.
• It would take too much energy for the cell to simply remove the phosphate from carbon 3 and reattach it to carbon 2 directly.

7. Enolase and PEP Formation

• 2-PGA --[Enolase; removes H2O]--> PEP (phosphoenolpyruvate)
• Enolase removes the OH on carbon 3 and the hydrogen on carbon 2 as water, forming a double bond with a phosphate on carbon 2.
• PEP is a very high-energy compound; delta G prime is minus 13.8 kcal/mol, more than enough to make an ATP.
• PEP's instability: if the phosphate were an OH group, it would spontaneously tautomerize from an enol to a more stable keto form. The phosphate group pins the molecule, preventing tautomerization until the next enzyme releases it.

8. Pyruvate Kinase and Completion of Glycolysis

• PEP + ADP --[Pyruvate kinase]--> Pyruvate + ATP (one-way reaction)
• At the pyruvate kinase active site, the phosphate is released and donated to ADP, allowing the keto-enol tautomerization to proceed, which provides a large negative delta G.

9. Lactate Dehydrogenase and NAD Regeneration

• Pyruvate + NADH + H+ --[Lactate dehydrogenase (LDH)]--> Lactate + NAD+
• The purpose of LDH is to regenerate NAD, because the pool size of NAD is very small. Without this step, NAD would not be available for the G3PDH reaction, and glycolysis would stop.
• Net yield from one glucose: 2 ATP.
• One role of glycolysis is to produce a small amount of ATP.

Exam emphasis: The lecturer stated students need to know the steps of glycolysis thoroughly.

10. Alcoholic Fermentation in Yeast

• Yeast perform alcoholic fermentation: glucose is processed through glycolysis to pyruvate, then the last two steps differ from the lactate pathway.
• Pyruvate --[Pyruvate decarboxylase; Mg2+ (cofactor), TPP (coenzyme)]--> Acetaldehyde + CO2 (one-way reaction)
• CO2 is nonpolar and can leave the yeast cell.
• Acetaldehyde --[Alcohol dehydrogenase]--> Ethanol + NAD+
• As with LDH in glycolysis, the purpose of alcohol dehydrogenase is to regenerate NAD because the pool size is small.
• Net ATP yield from alcoholic fermentation is also 2 ATP per glucose.

Exam emphasis: If asked to draw all steps of alcoholic fermentation, include the entire glycolysis pathway, not just the last two steps.

11. Michaelis-Menten vs. Allosteric Enzymes (Preview)

• Most glycolytic enzymes are Michaelis-Menten (hyperbolic velocity-vs.-substrate plot).
• Some key enzymes are allosteric (sigmoidal, S-shaped velocity-vs.-substrate plot); the lecturer indicated this would be discussed further.

12. NAD Structure and Spectral Properties

• NAD stands for nicotinamide adenine dinucleotide.
• The lower portion of NAD is adenosine monophosphate (AMP), which acts as a handle to help enzymes orient the molecule correctly in the active site. AMP handles are also found on coenzyme A, FAD, and other molecules.
• The business end is the nicotinamide ring (an amide, not an amine: C=O bonded to NH2).
• A hydride (H minus, carrying two electrons) is placed specifically at the top of the nicotinamide ring and nowhere else on the molecule.
• The positive charge on the nitrogen of NAD+ is partially delocalized by resonance to the carbon at the top of the ring, which attracts the hydride.
• Reduction of NAD+ to NADH eliminates the positive charge on the nitrogen.
• Students should be able to draw both the oxidized (NAD+) and reduced (NADH) forms.
• NADH absorbs light at 340 nm; NAD+ does not absorb at 340 nm. NAD+ absorbs at 260 nm; NADH also has slight absorbance at 260 nm, but the difference there is not useful.
• Biochemists set spectrophotometers to 340 nm to measure NADH specifically, because buffer, proteins, NAD+, and other substrates do not absorb at that wavelength.
• At 260 nm, proteins and nucleic acids absorb strongly, making that wavelength unsuitable for NADH measurement.

Reference figure · external source

13. Return to Allosteric Regulation of Glycolysis

• Generalization: reactions with large negative delta G values are typically catalyzed by allosteric enzymes with multiple subunits, showing sigmoidal kinetics.
• The allosteric enzymes in glycolysis are hexokinase, PFK1, and pyruvate kinase (all catalyze one-way reactions).
• In alcoholic fermentation, pyruvate decarboxylase is also allosteric (one-way reaction).
• Allosteric enzymes are much more sensitive to changes in compound concentrations in the cell.
• PFK1 regulation example:
  • ADP is a positive allosteric effector (activator): if ADP builds up, the cell is running low on energy and PFK1 speeds up.
  • ATP is a negative allosteric effector (inhibitor): if ATP builds up, the cell already has enough energy and PFK1 slows down.
  • ADP binds at a site separate from the active site, changes the enzyme's conformation, and increases its rate.
• Nothing in metabolism ever goes to zero; negative regulation means the rate slows down.

14. Purposes of Glycolysis

• Two main purposes:
  1. Produce some ATP (net 2 per glucose in both glycolysis and alcoholic fermentation).
  2. Provide intermediates that can be siphoned off for biosynthesis.
• Historical note: glycolysis was one of the first pathways discovered, which biased thinking toward phosphorylated compounds and coupled reactions as the universal mechanism for ATP production, which turned out to be incomplete.
• Biosynthetic examples:
  • 3-PGA can be siphoned off to make serine (S), which can be converted to glycine or cysteine.
  • DAP can be siphoned off to make glycerol, the backbone of phospholipids and triacylglycerols.
• Student question: Can 1,3-BPG be converted to 2,3-BPG? Answer (lecturer): Yes, in red blood cells. Red blood cells give up glycolysis to make 2,3-BPG (needed for hemoglobin function).

15. Equilibrium and Free Energy

• K-equilibrium is products divided by substrates at equilibrium.
• Cells never let reactions come to equilibrium; they operate in a steady state (open system). Biochemistry uses equilibrium math as a simplification because steady-state math is much more complicated.
• K-equilibrium prime accounts for situations where protons are involved in the reaction, measured at pH 7.
• Delta G = RT ln(products/substrates) minus RT ln(K_eq).
• If a system is at equilibrium, delta G = 0 and the reaction cannot do work. Cells cannot violate thermodynamics.
• If all species are at 1 molar, the ln(products/substrates) term becomes zero (ln(1) = 0), which is why 1 molar was chosen as the reference: it simplifies the math.
  • Under these conditions, delta G naught = -RT ln(K_eq).
• If all species are at 1 molar and pH is 7, the value is called delta G prime.
• The actual free energy available to a cell: delta G = delta G prime + RT ln(actual product-to-substrate ratio).
• Delta G prime values (like those given for 1,3-BPG and PEP) are reference values for comparing reactions in tables. The real delta G in a cell depends on the actual concentrations, which change throughout the day.

Exam emphasis: Equations will be provided on exams; students need to understand what the equations mean.

16. Free Energy Relationships

• Reversing a chemical reaction reverses the sign of delta G and inverts the equilibrium constant.
• Adding two chemical reactions (coupled reactions): delta G values are added, but equilibrium constants are multiplied.
• Application to come later in the course: the electron transport chain involves reactions flipping direction and changing the sign of delta G.
• The lecturer directed students to focus on the assigned example problems in Segal (homework one).

17. Beer's Law

• A = epsilon x L x C (absorbance = molar extinction coefficient x path length x concentration).
• Absorbance: the spectrophotometer measures I0 (initial light intensity) and I (light intensity after passing through the cuvette), then calculates log base 10 of (I0/I). Absorbance has no units.
• L (path length): distance from one face of the cuvette to the other; standard value is 1 cm.
• C (concentration): in molar (moles per liter); typically very small values in biochemistry.
• Epsilon (molar extinction coefficient): a large number with units M^-1 cm^-1 to cancel the units of C and L, since A is unitless. Example: 9,000 M^-1 cm^-1.
• Molecules must have conjugated double bonds to absorb light.
• Beer's Law is the equation of a straight line: A is directly proportional to C, and the plot passes through zero.
• Absorbance is proportional to concentration, not mass; the spectrophotometer does not integrate over volume. As long as the light beam is covered by solution, the reading is the same regardless of how full the cuvette is.
• Linearity must be verified experimentally; at high concentrations, the relationship curves off due to spectrophotometer limitations and intermolecular interactions.
• A practical test: dilute a sample in half and check that absorbance is halved.
• Student question: The lecturer acknowledged that changing pH, wavelength, or salt concentration can change the molar extinction coefficient, but stated this will not be explored further in BIS-103.

18. Alternative Beer's Law

• A = a x L x C, where little a is the specific absorbance.
• Used when the molecular weight of the absorbing species is unknown.
• Concentration is typically expressed in mg/mL or g/100 mL (the latter being a percent solution).
• Cannot calculate IUs using this form because IUs require micromoles per minute, which requires knowing the molecular weight.

19. LDH Enzyme Assay Using Beer's Law

• Reaction measured: Pyruvate + NADH + H+ --[LDH]--> Lactate + NAD+
• NADH absorbs at 340 nm; pyruvate, protons, LDH protein, and buffer do not absorb at 340.
• At time zero, absorbance at 340 is high (NADH present). If LDH is present, absorbance decreases over time as NADH is consumed.
• A flat line (no change) means no LDH is present.
• The initial linear region of the absorbance-vs.-time curve must be used (Beer's Law region). The slope of this region gives the initial velocity (v0).

20. IU Calculation for LDH

• From Beer's Law: if delta A changes per unit time, and epsilon and L are constants, then the concentration of NADH must be changing.
• Epsilon for NADH at 340 nm: 6,220 M^-1 cm^-1 (a known constant; would be given on an exam).
• Steps:
  1. Measure delta A per unit time from the linear region of the graph.
  2. Convert to a per-minute change (e.g., if measured over 15 seconds, multiply by 4).
  3. Calculate the change in NADH concentration (molar per minute) using Beer's Law.
  4. Multiply by the volume of the assay (in liters) to convert from moles per liter to moles. The liters cancel.
  5. Convert moles to micromoles (1 micromole = 1 x 10^-6 moles).
  6. The result equals the number of IUs of active LDH in the cuvette.
• Example: 0.15 micromoles of NADH consumed per minute = 0.15 IUs of active LDH.
• The NADH absorbance change is negative (NADH is consumed), but IUs are reported as a positive number.
• Units must be carried through calculations; guessing whether to multiply or divide leads to errors.

Exam emphasis: Homework 1 covers the IU calculation for the first midterm. Homework 2 and the final will extend the calculation further (e.g., determining IUs in the original extract).

21. First Midterm Logistics

• The first midterm covers material up through the Beer's Law and IU calculation material.
• Date: the Wednesday after next (not this Wednesday).
• An extra room has been confirmed in the Teaching and Learning Complex (TLC); details to be posted the next day.
• The answer key for homework 1 will be posted on Wednesday.

22. Transition: Starch and Glycogen

• Material from this point forward is on the second midterm.
• Animals and plants provide glucose by storing it as polymers: plants make starch, animals make glycogen.
• Both are very large polymers of glucose.

23. Starch Structure

• Amylose (~30% of starch by weight): a linear polymer of glucose residues linked by alpha-1,4 glycosidic bonds.
  • Alpha: the bond is below the plane of the sugar ring.
  • 1,4: the bond connects carbon 1 of one glucose to carbon 4 of the next.
  • OH groups must be drawn in the correct orientation: OH on carbon 2 is below the plane, carbon 3 above, carbon 4 below.
  • The OH on carbon 1 is below the plane when part of a polymer because an enzyme links it to the next residue.
  • Can contain hundreds or more glucose residues; each linkage required energy to form.
• Amylopectin (~70% of starch by weight): a branched polymer of glucose.
  • Branches occur approximately every 24 residues (not precisely known how the enzyme determines this spacing).
  • Branch points involve alpha-1,6 linkages: carbon 1 of the branch glucose is linked to carbon 6 of a glucose in the main chain, with the bond below the plane of the donating glucose.

24. Reducing and Non-Reducing Ends

• Reducing end: the terminal glucose whose anomeric carbon can open and close (hemiacetal); the aldehyde form can reduce metal ions. Only one per amylose molecule.
• Non-reducing end: terminal glucoses whose anomeric bonds cannot open and close (acetal); protected from oxidation.
• All internal glucose residues are also non-reducing (protected).
• In a polymer of 400 residues, 399 are protected from oxidation; only the one reducing end is vulnerable.
• Branching creates additional non-reducing ends, increasing the number of sites where glucose can be added or removed.
• All chemistry of adding or removing glucose residues in both starch and glycogen occurs at non-reducing ends.
• Amylose (linear) has only one non-reducing end per molecule, making it less efficient for rapid glucose addition or removal compared to amylopectin.

25. Glycogen Structure and Function

• Glycogen is the animal storage form of glucose.
• Stored primarily in the liver; enough glycogen for about one day of glucose supply.
• Glycogen is constantly being made and demolished as needed (e.g., the brain requires glucose).
• Glycogen is more branched than starch: branches occur approximately every 8 residues.
• More branches mean more non-reducing ends, allowing animals to add or remove glucose residues at many points simultaneously.
• Adaptive advantage: animals (e.g., fleeing a predator) need rapid mobilization of glucose, which benefits from the many non-reducing ends in glycogen.

Reference figure · external source

26. Glycogenin: Initiating Glycogen Synthesis

• A fully formed glycogen molecule contains about 50,000 glucose residues.
• Glycogen synthesis begins with glycogenin, a small dimeric protein (two identical subunits, quaternary structure).
• Each subunit has an active site. It is not known whether the two active sites cooperate or function independently.
• Each active site contains a tyrosine residue whose side chain hydroxyl group (benzene ring-CH2-OH) serves as the attachment point for glucose.
• Glycogenin uses UDP-glucose (uridine diphosphate glucose) as the activated glucose donor. UDP is released as a product in a coupled reaction that provides the energy for covalent bond formation.
• The uridine portion of UDP-glucose acts as a zip code, directing the molecule to specific enzymes. Other nucleoside diphosphates direct molecules to other enzymes.
• Student question: Which hydroxyl on glucose is linked to the tyrosine? Answer (lecturer): the OH on carbon 1.
• Glycogenin adds a total of 8 glucose residues, linked in alpha-1,4 linkages (including the first glucose to the tyrosine).
• This 8-mer acts as a primer for the next enzyme.
• Glycogenin is both the enzyme that builds the primer and the anchor of the growing glycogen molecule. Whether it remains attached throughout the life of the glycogen molecule or detaches to start new ones is unknown.

27. Glycogen Synthase

• Glycogen synthase is a completely different enzyme from glycogenin.
• It requires the 8-mer primer built by glycogenin; it cannot initiate synthesis on its own.
• It uses UDP-glucose as the substrate and releases UDP as a product.
• It extends the chain one glucose at a time in alpha-1,4 linkages and can add hundreds of residues.
• Naming convention: synthase (does not use a nucleoside triphosphate) vs. synthetase (uses a nucleoside triphosphate like ATP).
• General principle: polymerases need to build off of something (a primer), and this requirement is a common control mechanism in cells.

28. Closing Announcements

• The lecturer encouraged students to start homework 1.
• All glycogen material is designated for the second midterm.
• Homework 2 (covering second midterm material) will be posted sometime early the following week.