Microcalorimetry and Introduction to Glycolysis

Lecture Overview

This lecture covers microcalorimetry as the third technique for studying cell metabolism, following earlier discussions of other analytical methods. The lecturer explains the instrument setup, experimental protocol (Parts A, B, and C), and how heat, CO2 production, and oxygen consumption are quantified from living tissue samples. The second half introduces key concepts of intermediary metabolism, including catabolic versus anabolic pathways, coupled enzyme reactions (illustrated through glutamine synthetase), and the first four steps of glycolysis through the aldolase reaction.

Key Concepts and Definitions

• Microcalorimetry: Measurement of very small flows of heat from living cells, operating in the microwatt range; distinct from freshman chemistry calorimetry, which burns compounds to completion.
• Gibbs free energy equation: Delta G = Delta H - T Delta S; Delta G is the energy available for work, Delta H is change in enthalpy/heat, T is temperature in Kelvin, Delta S is entropy/disorder.
• Open system: A system with energy flowing in and out; living cells are open systems in steady state.
• Steady state: Energy flowing in and flowing out at balanced rates; important to living cells and enzyme kinetics.
• Intermediary metabolism: The study of small organic compounds (~1,000 molecular weight or less) such as ATP, glucose, fatty acids, and amino acids.
• Catabolic reactions: Reactions that break down compounds; converge on acetyl-CoA.
• Anabolic reactions: Reactions that build compounds; diverge/branch from common precursors.
• Acetyl-CoA: A two-carbon acetyl group carried by coenzyme A (CoA) via a thioester bond (sulfur connected to a carbonyl group); the convergence point of catabolic pathways.
• Coupled reaction: Two reactions physically linked at an enzyme's active site so that an exergonic reaction drives an endergonic one.
• Acyl phosphate: A phosphate attached to a carbonyl group; a high-energy compound (large negative Delta G).
• Glycolysis: The lysis (breakage) of sugars; occurs in the cytosol; breaks glucose down to pyruvate/lactate.
• Glucose transporter: A 45,000 MW membrane protein that facilitates diffusion of glucose across the plasma membrane; humans have 14 forms (GLUT1-14).
• Domain: Local tertiary structure within a protein.

Chronological Lecture Notes

1. Course Business

• The lecturer expects to finish midterm 1 material by the end of Monday.
• Today is equivalent to a Friday in the schedule; students encouraged to introduce themselves to neighbors for study partnerships.

2. Introduction to Microcalorimetry

• Microcalorimetry is the third technique for examining cells, focused on measuring the flow of heat from living cells.

• This differs from freshman chemistry calorimetry, where a substance is burned to completion and total heat released is measured. In microcalorimetry, the cells remain alive, and very tiny flows of heat are detected.

• The instrument can be modified to measure the rate of CO2 production and the rate of oxygen consumption.

• Eukaryotic cells produce a lot of CO2, mostly from mitochondria. Mitochondria are also the principal organelles consuming oxygen.

• The Gibbs free energy equation (always provided on exams) describes the energy a system can use for work:

  Delta G = Delta H - T Delta S

  • Delta G: free energy available for work
  • Delta H: change in heat/enthalpy
  • T: temperature in Kelvin
  • Delta S: entropy/disorder

• The microcalorimeter measures only Delta H.

Exam emphasis: The lecturer always provides the Gibbs free energy equation on exams and expects students to understand its concepts, not calculate with it initially.

3. Scale of Measurement and LED Analogy

• An LED night light (0.7 watts = joules per second) was used to illustrate the scale.
• The light bulb is an open system: energy flows in, energy flows out (mostly as light, a little as heat). This is a steady state.
• Dividing the light output repeatedly by factors of ten (first by one-seventh, then tenths) demonstrates the realm where the microcalorimeter operates: around one microwatt.
• Human senses and other lab instruments (spectrophotometer, liquid scintillation counter) cannot detect heat at this scale; the microcalorimeter is required.
• Professor Richard Criddle on this campus was the expert on microcalorimetry; the lecturer worked with him.

4. Instrument Setup: the Ampule

• The ampule is a small metal container roughly thumb-sized, with a volume of about one milliliter.
• Made of a special metal alloy excellent at transferring heat.
• A small metal cylinder is welded to the ampule interior, creating a well where approximately 40 microliters of liquid can be pipetted.
• Tissue (plant leaves, bacteria, red blood cells, etc.) is placed inside the main chamber; the tissue must not contact the liquid in the upper well.
• A metal lid screws on tightly. A small hole in the lid is fitted with thin tubing (fiber optic tubing) connected to a pressure sensor.
• The tubing is very thin to prevent heat from outside the ampule from conducting in.

5. Instrument Setup: the Microcalorimeter

• A top-view of the instrument shows four openings: one reference position (empty ampule, never changed) and three sample positions (ampules 1, 2, and 3).
• Each position is surrounded in three dimensions by heat sensors that convert heat into electrical signals sent to the computer on separate channels.
• The ampule fits snugly so that heat from cells passes through the metal walls to the sensors.
• Example experimental design: ampule 1 might contain 10 leaves of a normal plant; ampule 2 might contain 10 leaves of a plant treated with a pesticide, to test whether the pesticide changes plant metabolism.
• Individual lids are placed on each ampule position, then a large lid covers the entire instrument, all to keep lab heat from interfering. Room temperature causes heat sensors to go off-scale.

6. Experimental Protocol: Part A (Baseline Heat Rate)

• On the y-axis: microwatts (joules per second) per milligram of dry weight. On the x-axis: time in minutes.
• Data are normalized to dry weight because the amount of tissue varies between ampules. Dry weight is used instead of cell count for tissues like leaves.
• When ampules are first inserted, heat is off-scale due to room heat entering; the instrument recovers and the signal falls, eventually leveling off.
• The plateau value is the heat flow from the living cells. A real numerical value is obtained (e.g., ~80 microwatts).
• If the signal levels off at zero, the cells are dead.
• Part A typically runs for about 30 minutes with 40 microliters of water in the upper well.

7. Experimental Protocol: Part B (CO2 Trap with NaOH)

• After Part A, each ampule is removed, the 40 microliters of water is replaced with 40 microliters of 0.4 M sodium hydroxide (a strong base, deliberately in excess).
• The NaOH sits in the upper well and never touches the tissue.
• The instrument recovers from being opened, then the signal levels off at a higher plateau than Part A.
• The difference between the Part A and Part B plateaus is proportional to the CO2 production rate.
• NaOH acts as a CO2 trap: OH- attacks the partial-positive carbon of CO2 (both oxygens pull electron density away from carbon), forming bicarbonate. Excess OH- then converts bicarbonate to carbonate. These reactions are exothermic.
• CO2 is nonpolar and passes through cell membranes freely. Inside the sealed ampule, CO2 molecules bounce at high velocity (ideal gas behavior). When a CO2 molecule hits the NaOH surface, it is chemically trapped as carbonate and releases heat that the instrument detects.
• The heats of reaction for this chemistry are well known; using those values, the measured heat can be converted to nanomoles or picomoles of CO2 produced per minute.
• NaOH is in excess to ensure every CO2 molecule is captured.
• If Part B levels off at the same value as Part A, no detectable CO2 is being produced, which for plant cells would indicate nonfunctional mitochondria.

8. Experimental Protocol: Part C (Control)

• After Part B (~30 minutes), each ampule is removed, NaOH is replaced with 40 microliters of water.
• The signal should return to approximately the Part A baseline.
• Part C is a control: it verifies that nothing harmful happened during Part B (e.g., NaOH accidentally dripping onto tissue, which would cause heat from cell destruction unrelated to CO2).
• Without Part C, one would have to assume Part B caused no damage, which is undesirable in science.

9. Reasons for Water in Part A

• Control for water vapor: the NaOH solution in Part B is mostly water. Water in Part A controls for the possibility that water vapor itself changes cell metabolism, ensuring the Part B effect is due to NaOH specifically.
• The second reason was deferred to the next slide/topic.

10. Dry Weight Determination

• After the experiment (~90 minutes total), the tissue is removed and placed into individual containers.
• Samples are dried in a vacuum oven at 90 degrees Celsius overnight.
• The vacuum prevents oxidation (burning) of the tissue, which would reduce the dry weight.
• Each dried sample is weighed on a precise balance; this is the dry weight used to normalize the heat data.

11. Pressure Sensor and Oxygen Measurement

• Pressure data are collected simultaneously with heat data throughout the experiment, from sensors connected to each ampule.
• In Part A and Part C, multiple gases (nitrogen, oxygen, CO2) contribute to pressure, so individual gas contributions cannot be separated.
• In Part B, NaOH eliminates CO2, and nitrogen is inert, so any pressure decrease is attributable to oxygen consumption, mainly by mitochondria.
• Picomoles of oxygen consumed per minute can be calculated using PV = nRT (the ideal gas law: pressure times volume equals n times gas constant times temperature).
• A second y-axis (pressure) can be added to the same plot to show the pressure decrease during Part B.

12. Summary of Microcalorimetry Output

• Three quantities are obtained with real numerical values:
  1. Heat rate per milligram dry weight
  2. Rate of CO2 production
  3. Rate of oxygen consumption
• These metabolic measurements cannot be obtained from DNA microarrays or 2D-IEF-SDS-PAGE gels.

13. Application: Predicting Tree Growth

• Microcalorimetry can determine in two to three days, from small tissue samples, which tree varieties have the best metabolism and growth potential, compared to years of qualitative observation in the field.
• The measurements are highly predictive of outcomes 10 years later.
• Also applicable to cancer cells and other systems.
• Samples do not need to be very pure; rinse off debris, avoid contamination with other organisms, and run many replicates (~20) for averaging.

14. Key Concept: Intermediary Metabolism

• The course studies intermediary metabolism: small organic compounds of approximately 1,000 molecular weight or less (ATP, glucose, fatty acids, amino acids, etc.), not large macromolecules like proteins, DNA, or RNA.
• Catabolic reactions break down compounds. Anabolic reactions build compounds.
• Energy-carrying molecules connect catabolic and anabolic pathways:
  • NAD (nicotinamide adenine dinucleotide) and its reduced form NADH
  • NADP (phosphorylated form of NAD) and NADPH
  • FAD (flavin adenine dinucleotide) and FADH2
  • ATP
• These compounds exist in small pool sizes. Cells do not store large quantities of ATP, NADH, or FADH2.
• To build something (requiring ATP or other energy carriers), a cell must simultaneously break something down, and vice versa. The two types of pathways are perfectly meshed.

15. Types of Metabolic Pathways

• Convergent (catabolic): Many substrates (glucose, glycogen, fatty acids) funnel into a single compound, acetyl-CoA. This is efficient, analogous to multiple roads funneling into one freeway (Interstate 80 analogy) rather than building parallel pathways.
• Divergent (anabolic): Pathways branch out like tree branches for biosynthesis.
• Cyclic: Intermediates are recycled. In the Krebs cycle, two carbons enter as acetyl-CoA and two carbons leave as CO2, so only tiny amounts of intermediates are needed; in theory, the cycle could run infinitely.
• Course plan: glycolysis (in the cytosol) for midterm 1; Krebs cycle (in the mitochondria) for midterm 2.

Exam emphasis: Students must know glycolysis and the Krebs cycle thoroughly; the lecturer compared the expectation to knowing them "like the back of your hand."

• Acetyl-CoA: an acetyl group (two carbons) carried by coenzyme A as a thioester (sulfur connected to a carbonyl group).

16. Break

17. Amino Acid Review: Glutamate and Glutamine

• The lecturer presented a structure for students to identify: glutamic acid (glutamate when charged). One-letter code: E.
• Glutamate reacts with an ammonium ion to form glutamine. One-letter code: Q.
• Students are expected to retain amino acid structures and one-letter codes from BIS-102 for their careers in biological sciences and medicine.

18. Coupled Enzyme Reactions: Glutamine Synthetase

• Ammonium ions at high concentration are toxic to cells. Many nitrogen-containing compounds (proteins, DNA, RNA) are broken down to ammonia in metabolism, which becomes ammonium at cell pH.

• Glutamate + ammonium ion --> glutamine removes the toxic ammonium.

• This reaction is endergonic and does not proceed well on its own.

• Cells couple this endergonic reaction with the exergonic breakdown of ATP at the active site of one enzyme, glutamine synthetase.

• The coupled reaction proceeds in one direction:

  Glutamate + NH4+ + ATP --[Glutamine synthetase]--> Glutamine + ADP + Pi

• Some amino acids (like glutamate) serve not only as protein building blocks but also to detoxify compounds.

19. Mechanism of Glutamine Synthetase

• Step 1: ATP phosphorylates the side-chain carboxyl group of glutamate (never the alpha-carboxyl), producing an acyl phosphate intermediate and ADP. The enzyme orients glutamate so that only the correct carboxyl is phosphorylated.
  • Acyl phosphate: a phosphate on a carbonyl group (C=O with O-phosphate).
  • Acyl phosphates are high-energy compounds (large negative Delta G).
  • There is an acyl phosphate intermediate in glycolysis as well.
• Step 2: The ammonium ion diffuses into the active site. A group at the active site removes a proton, converting ammonium to ammonia. The nitrogen lone pair of ammonia acts as a nucleophile, attacking the carbonyl carbon of the acyl phosphate (the carbon is electrophilic because surrounding oxygens withdraw electron density). This displaces the phosphate group, producing glutamine and inorganic phosphate (Pi).

Exam emphasis: Acyl phosphates are high-energy compounds; students should recognize them immediately (a "red flag").

20. Historical Note on Coupled Reactions

• Coupled reactions (such as the glutamine synthetase mechanism) were discovered early in biochemistry, largely through the study of glycolysis.
• This led to the incorrect assumption that all cellular energy-transfer reactions work by substrate-level coupling.
• The later discovery of proton gradients revealed that cellular energy metabolism is more complicated.
• Coupled reactions do not have to involve ATP; they can involve other compounds.

21. Glycolysis: Overview and Strategy

• Glycolysis means the lysis (breakage) of sugars. The course focuses only on glucose.
• Glycolysis occurs in the cytosol.
• The pathway goes from glucose all the way down to lactate. Alcoholic fermentation (in organisms like yeast) diverges at pyruvate, going to ethanol instead.
• The cell's strategy: phosphorylate glucose at carbon 6, phosphorylate at carbon 1, then split the six-carbon molecule down the middle to produce two three-carbon fragments, each with a phosphate.
• Topics to cover: reactions, regulation, and purpose.

Exam emphasis: Homework 1 requires learning the full glycolysis pathway. Glycolysis material is on midterm 1.

Reference figure · external source

22. Glucose Structure

• Beta-D-glucose has six carbons. Carbon 1 is at the anomeric position.
• Shorthand notation: vertical lines represent hydroxyl (OH) groups.
  • C2: OH below the plane of the ring
  • C3: OH above the plane
  • C4: OH below the plane
• C1 OH can be above (beta form) or below (alpha form) because the bond between the ring oxygen and C1 is labile; the ring opens and closes (mutarotation).
• The beta form (OH above) is slightly more stable in solution.
• Drawing C1 OH as a horizontal line indicates it could be either above or below.

23. Glucose Transport Across the Membrane

• Glucose is very polar due to multiple oxygen atoms. Polar atoms: oxygen, nitrogen, sulfur. Charged molecules are even more polar.
• The interior of the plasma membrane is nonpolar; glucose cannot cross without help from a protein.
• The glucose transporter is a membrane-integrated protein of 45,000 molecular weight that facilitates diffusion of glucose in both directions.
• X-ray crystallography of this protein is not available because membrane-integrated proteins are difficult to extract and crystallize.
• Humans have 14 different glucose transporter genes (GLUT1 through GLUT14), reflecting the importance of glucose and differing tissue requirements. The brain runs mostly on glucose.

24. Glycolysis Step 1: Hexokinase

• Glucose --[Hexokinase; Mg2+; ATP]--> Glucose-6-phosphate + ADP

• Hexokinase phosphorylates the C6 hydroxyl group specifically (not any other OH), coupled with ATP breakdown.

• "Hexokinase" implies it can handle other hexose sugars, not just glucose.

• At physiological pH, the phosphate on C6 carries a minus-2 charge (P=O, O-, O-). Students may write a circled P on exams.

• This step consumes one ATP, even though a purpose of glycolysis is to produce ATP.

• Accomplishments of this step:

  1. Pulls the equilibrium: glucose disappears (becomes G6P), allowing more glucose to enter the cell.
  2. Traps glucose inside the cell: the glucose transporter will not accept glucose-6-phosphate because it is a different molecule.
  3. Puts a charged "handle" on C6 that helps the next enzyme orient the substrate at its active site.
  4. Raises the energy of glucose slightly by transferring some energy from ATP.

• Hexokinase requires magnesium (Mg2+) as a cofactor. It will not work with zinc, manganese, or other metal ions.

25. Role of Magnesium in Hexokinase

• ATP at physiological pH carries many negative charges.
• The phosphorus atom targeted by the C6 hydroxyl has a partial positive charge (surrounding oxygens withdraw electron density).
• The C6 hydroxyl (partial negative, acting as nucleophile) cannot easily approach through the cloud of negative charges on ATP.
• Mg2+ (plus-2 charge) sits near the phosphates of ATP, shielding/dampening the negative charges so the nucleophilic OH can approach and attack the phosphorus.
• Without Mg2+, the OH does not attack.

26. Hexokinase Conformational Change and Water Exclusion

• The attack by the C6 hydroxyl is 40,000 times faster than attack by water.
• X-ray crystallography of hexokinase from yeast (a dimer; one subunit shown) revealed the explanation.
• The enzyme has two domains. Without glucose bound, the domains are in an open conformation.
• When glucose binds, it triggers a conformational shift: the two domains close like a clamshell, burying glucose and physically excluding water.
• This explains why water is not a significant competing nucleophile.
• The enzyme with only glucose bound (no ATP) cannot catalyze the reaction, enabling crystallographic study of the glucose-bound state.

Reference figure · external source

27. Glycolysis Step 2: Phosphoglucose Isomerase (PGI)

• Glucose-6-phosphate --[PGI]--> Fructose-6-phosphate

• PGI moves the bond from the ring oxygen down to carbon 2, converting carbon 1 from an aldehyde to an alcohol (OH).

• An aldose is converted to a ketose.

• The purpose: carbon 1 in the aldehyde form is an electrophilic target, not a nucleophile. Converting it to an alcohol (OH) allows it to attack ATP in the next step.

• This reaction is totally reversible.

28. Glycolysis Step 3: Phosphofructokinase-1 (PFK1)

• Fructose-6-phosphate + ATP --[PFK1]--> Fructose-1,6-bisphosphate + ADP

• PFK1 phosphorylates the C1 hydroxyl using a second molecule of ATP.

• At this point, glycolysis has consumed two ATPs.

• PFK1 is one-way (irreversible).

• A second isomer, PFK2, exists and will be discussed later in the course.

29. Glycolysis Step 4: Aldolase

• Fructose-1,6-bisphosphate --[Aldolase]--> Dihydroxyacetone phosphate (DAP) + Glyceraldehyde-3-phosphate (G3P)

• Aldolase splits fructose-1,6-bisphosphate between carbons 3 and 4, generating two three-carbon molecules, each with a phosphate.

30. Closing and Preview

• The remainder of glycolysis will be covered on Monday.
• The end of Monday's lecture will define the boundary of midterm 1 material.
• Students were urged to learn the pathway over the four-day holiday.