BIS 103 Course Introduction and Metabolism Overview

Lecture Overview

This opening lecture introduces BIS 103, a six-week summer course on intermediary metabolism. Dr. Hilt outlines the exam structure, grading policy, and course themes — weak bonds, entropy, metal ions, and proton gradients — before surveying how cells function as chemical engines governed by thermodynamics. Three techniques for studying overall cellular metabolism (DNA microarrays, 2D gels, microcalorimetry) are introduced, and the mitochondrion and chloroplast are previewed as major organelles for the final third of the course. The second half of the lecture reviews BIS 102 foundations: buffer chemistry, the Henderson-Hasselbalch equation, NADH and the pyruvate-to-lactate reaction, Michaelis-Menten versus allosteric enzyme kinetics, and the beginning of affinity chromatography.

Key Concepts and Definitions

• Intermediary metabolism: The biochemical reactions occurring in living cells.
• Assay: A particular way of measuring something; for enzymes, it measures how much product is made per unit time.
• ΔG°' (delta G prime): Standard free energy change with all reactants at 1 M except protons at 10⁻⁷ M (pH 7); used to compare chemical reactions fairly.
• NADH: Nicotinamide adenine dinucleotide, reduced form; carries two electrons as a hydride (H⁻).
• NAD⁺: Oxidized form of nicotinamide adenine dinucleotide.
• Hydride (H⁻): A hydrogen with two electrons; one of three oxidation states of hydrogen (H⁺, H·, H⁻).
• Vmax: Maximum velocity of an enzyme; characterizes how fast the enzyme can go when saturated with substrate.
• Km (Michaelis constant): A measure of how well an enzyme binds its substrate.
• Turnover number: The number of substrate molecules one enzyme molecule converts to product per minute; approximately 30,000 for most enzymes.
• KI: Equilibrium dissociation constant of an inhibitor; describes how well an inhibitor binds to an enzyme.
• Michaelis-Menten enzyme: An enzyme producing a hyperbolic velocity-versus-substrate curve.
• Allosteric enzyme: An enzyme producing a sigmoidal velocity-versus-substrate curve; typically has cooperating subunits.
• Cooperativity (n): Exponent in the allosteric enzyme rate equation measuring cooperativity among subunits; a larger n produces a steeper sigmoidal curve.
• Transcriptome: The complete set of messenger RNAs a cell is producing at a given time; studied via DNA microarrays.
• Proteome: The complete set of proteins a cell is producing; studied via two-dimensional gel electrophoresis.
• Henderson-Hasselbalch equation: pH = pKa + log([B]/[A]), relating pH to the ratio of base form to acid form of a buffer.
• Tris: A man-made buffer with three CH₂OH groups and one amino group; pKa approximately 8.2; acid form carries +1 charge, base form is neutral.
• Affinity column: A protein purification method exploiting biological specificity; uses a polar bead matrix with a covalently attached ligand.
• Pyruvate: A three-carbon compound; C1 is a carboxyl group (pKa 2.5); exists in the deprotonated base form at pH 7.
• Lactate dehydrogenase (LDH): Enzyme catalyzing the reversible reaction of pyruvate + NADH + H⁺ to L-lactate + NAD⁺; the last step of glycolysis.
• Microcalorimetry: A technique measuring very small amounts of heat produced by living cells; can also determine rates of CO₂ production and O₂ consumption.

Chronological Lecture Notes

1. Course Introduction and Staff

• Lecturer is Dr. Hilt.
• Two learning assistants: Cat and Wiley. Both have previously taken BIS 103, hold office hours, and receive one unit of MCB 197T for three hours per week.
• Students interested in becoming a learning assistant in the upcoming academic year can email Dr. Hilt (klhilt@ucdavis.edu).
• Dr. Hilt will teach two large sections of BIS 105 in winter; BIS 105 is half BIS 102 and half BIS 103.

2. Textbook and Course Scope

• Required textbook: a paperback workbook written by Erwin Sagal, a retired faculty member of the department; special rate available.
• The workbook helps with calculations important in biochemistry and is useful in a research lab setting.
• Three main areas covered using the workbook:
  1. Weak acids and bases — pH affects metabolism; cells use buffers; pH affects hydrogen bonds and ionic bonds.
  2. Enzyme kinetics — enzymes catalyze virtually all cellular reactions; most enzymes are proteins.
  3. Energetics — cells must capture and utilize energy from consumed materials to build proteins, DNA, cell walls, etc.
• The course focuses on intermediary metabolism: the biochemical reactions in living cells.

3. Exam Structure and Grading

• Two midterms and one final exam.
• The ten-week course is compressed into six weeks (meets Monday, Tuesday, Wednesday only).
• First midterm is pushed back to week three.
• No final exam week in summer session; the final is on the last day of class.
• Each midterm is 50 minutes; no lecture after a midterm day.
• The final is one hour and 50 minutes; most students finish in about one hour.
• Question types: calculation, short answer, some multiple choice (limited); no Scantron.
• Bring a calculator; all equations and constants are furnished.
• Exams test on lecture content and homework.
• Homework 1 will be posted the following day; it is not graded.

Exam emphasis: "You're crazy if you don't do the homework."

• Final exam is worth 200 points (twice the value of a midterm).
• Half of the final covers material after the midterm 2 cutoff (like a third midterm); the other half covers material from midterms 1 and 2.
• Grading is on a curve: the class average is the lowest B-minus; the top approximately 15% receive some form of A.

4. Course Content Overview

• The course studies metabolism — all the reactions in a cell — though not all can be covered.
• Emphasis on understanding overall metabolism rather than studying pathways in isolation; all pathways are tied to each other.
• Three techniques for viewing overall cellular metabolism will be explored.
• The first pathway studied will be glycolysis.
• Amino acid biosynthesis pathways are usually very complicated and reserved for graduate courses; BIS 103 focuses on core pathways that are broadly considered essential.
• Pathways usually serve more than one function.
• Enzyme activity measurement requires an assay: measuring how much product is made per unit time.
• Every step in a pathway is catalyzed by an enzyme; enzymes must be controlled (inhibited or activated) so they do not convert substrate unchecked.
• Reactions with large negative ΔG values especially need tight control; without regulation, the enzyme would convert all available substrate.

5. MCB 120L Lab Course Recommendation

• MCB 120L is a lecture-lab course in Molecular and Cellular Biology; labs are on the third floor of Briggs Hall.
• Strongly recommended: doing lab work reinforces BIS 102 and BIS 103 content through hands-on experience and learning from mistakes.
• Beneficial preparation for MCATs and professional school.

6. Course Themes

• Weak bonds: Four types of weak (non-covalent) bonds are important throughout biochemistry and metabolism. They form and release, allowing enzymes to recognize specific substrates at active sites through multiple weak bonds; they are also central to protein structures, membranes, and double-stranded DNA.
• Entropy (ΔS): Change in entropy is an important concept throughout the course.
• Metal ions: Certain enzymes require specific metal ions at their active sites to function. Without the correct metal ion, the enzyme does not convert substrate to product. Metal ion specificity is strict — for example, an enzyme requiring Mg²⁺ will not function with Mn²⁺ or Cu²⁺ because the atomic orbitals differ. Research has shown that some proteins position two different metal ions next to each other to carry out necessary chemistry. Normal dietary intake provides sufficient metals.
• Proton gradients across membranes: Transporting protons across membranes requires energy. In mitochondria, the electron transport chain (ETC) creates a proton gradient critical for ATP production. In chloroplasts, photosystems 1 and 2 create proton gradients during the light reactions of photosynthesis. Proton-gradient-driven ATP synthesis is how most ATP is made on the planet for all organisms.

7. The Cell as a Chemical Engine

• An animal cell is an open system: nutrients flow in (e.g., glucose, O₂), waste products flow out (CO₂, water, heat/entropy).
• If a cell cannot take in nutrients, it cannot maintain its ordered structure or carry out necessary reactions.
• Cells cannot accumulate waste products.
• Water is the major component of any cell by weight; forming water is beneficial.
• No living cell harnesses all consumed energy; some is always lost as heat and entropy.
• A cell is a chemical engine that obeys the laws of thermodynamics.
• ΔG = free energy available to a cell to perform work (movement, division, biosynthesis).
• ΔH = change in heat or enthalpy.
• T = temperature in Kelvin.
• ΔS = entropy, a measurement of disorder.
• Glucose (a six-carbon, highly ordered compound) is broken down into six CO₂ molecules (separate, random, able to leave the cell).
• ΔG°' for glucose breakdown to CO₂ and water = −686 kcal/mol (a large number; not to be memorized).
• ΔG°' for ATP synthesis from ADP + Pᵢ = +7.2 kcal/mol (positive because energy input is required).
• Inorganic phosphate (Pᵢ): structure is phosphorus double-bonded to one oxygen, with two O⁻ groups and one OH, around pH 7.
• There is plenty of free energy in glucose to drive the synthesis of many ATP molecules.
• The course uses kcal/mol (following the Sagal textbook); newer textbooks use joules. Calories can be converted to joules by multiplying by a constant.
• Everything in the cell is dynamic: mitochondria move toward and away from the nucleus, fuse, and divide.
• Systems biology: everything in the cell communicates; changing the ATP concentration affects many processes.
• All studied pathways occur simultaneously and are interconnected.
• Quantum mechanics applies to protons, electrons, and photons in cells; quantum biology is an emerging frontier that will deepen understanding in coming years.

8. Diverse Cell Types and Their Common Features

• Plant cells: photons of light hit chloroplasts; how chloroplasts work is the last topic in the course.
• Bacteria (e.g., E. coli): very small, visualized with electron microscopy; widely used in biochemistry research. DNA fragments (e.g., a human gene) can be inserted into bacteria to produce large quantities of a protein for study.
• Commonalities across animal cells, plant cells, and bacteria:
  • All use DNA as their genetic code.
  • Some pathways are shared (e.g., glycolysis).
  • All obey the laws of thermodynamics.
  • All are dynamic.
  • Mitochondria and chloroplasts are believed to have evolved from bacteria.

9. Enzyme Kinetics Concepts and Assays

• Kinetics: how fast a reaction occurs. Thermodynamics: the potential of a reaction to occur. A reaction with a large negative ΔG is thermodynamically favorable but without an enzyme has no chance kinetically.
• Vmax: characterizes how fast an enzyme can go.
• Km (Michaelis constant): mathematically describes how well an enzyme binds its substrate.
• Turnover number: for one molecule of enzyme, how many substrates are converted to product in one minute; approximately 30,000 for most enzymes. This means cells do not need to build many copies of a given enzyme.
• KI: equilibrium dissociation constant of an inhibitor; the inhibitor binds somewhere on the enzyme and slows it down.
• Assays are required to follow enzyme activity. Methods include spectrophotometry, radioisotopes, and fluorescence.
• Coupled enzyme assays: when the substrate/product of interest does not absorb light, additional enzymes and compounds can be added so that a light-absorbing species connected to the enzyme of interest is measured instead.
• Understanding the chemical mechanism at the active site (which groups do what chemistry) has practical applications: knowing how an enzyme works can lead to designing compounds that inhibit it, potentially saving lives (e.g., enzymes involved in cancer).
• Medicine is fundamentally about salvaging disrupted metabolism; every disease involves something wrong with one or more enzymes.

10. Energetics in Metabolism

• In biochemistry: ΔG (free energy), ΔH (enthalpy), ΔS (entropy), and ΔE (change in reduction potential, relevant to redox reactions).
• Most reactions in metabolism involve a flow of electrons: one compound gives up electrons, another accepts them. This is measured as ΔE.

11. Applications of Studying Metabolism

• Medicine: diseases are complicated disruptions of metabolism; simple ones would already be solved.
• Research: fundamental discoveries can blossom into important applications years later.
• Crop improvement: as global population grows, engineering plants (e.g., inserting genes for atmospheric nitrogen fixation) could reduce dependence on fertilizer.
• Organism relatedness: many metabolic pathways are shared across diverse organisms; studying an unfamiliar but experimentally tractable organism can yield discoveries relevant to human metabolism.

12. Three Techniques for Overall Cellular Metabolism

• DNA microarrays (transcriptome):

  • Determine which messenger RNAs a cell is making at the time of investigation.
  • Always used comparatively (e.g., cancer cells vs. normal cells).
  • Extract all mRNAs and look for differences.
  • Homework 1 includes an online interactive problem on DNA microarrays.

• Two-dimensional (2D) gel electrophoresis (proteome):

  • First dimension: isoelectric focusing (IEF) — separates proteins by isoelectric point (pI).
  • Second dimension: SDS-PAGE — separates proteins by subunit size.
  • Powerful method for comparing all proteins being made (e.g., cancer vs. normal cells).
  • Can detect changes that DNA microarrays miss: a single base-pair change in mRNA might replace a valine (no charge on R group) with a glutamate (charged R group), shifting the protein's position on the 2D gel.

• Microcalorimetry:

  • An instrument that measures very tiny amounts of heat given off by living cells.
  • With proper setup, can also measure the rate of CO₂ production and O₂ consumption.
  • O₂ consumption is a measure of mitochondrial function (most inhaled O₂ goes to mitochondria in eukaryotes).

• Using multiple techniques provides different perspectives on the same biological question; relying on a single method can be misleading.

13. Mitochondria and Chloroplast Preview

• Mitochondria (focus of the last third of the course):

  • Pyruvate dehydrogenase (PDH) complex.
  • Krebs cycle — enzymes in the matrix (inner sanctum).
  • Fatty acid breakdown — also in the matrix.
  • Electron transport chain (ETC) — thousands of copies embedded in the inner membrane.
  • ATP synthase — also embedded in the inner membrane; the machine that makes most of the ATP on the planet.
  • Bacteria lack mitochondria but have ATP synthases embedded in their inner membranes.

  Reference figure · external source

• Chloroplasts (last topic of the course):

  • Photosynthesis occurs in thylakoid membranes via photosystems 1 and 2 (light reactions).
  • The stroma (liquid surrounding thylakoid membranes) contains the enzymes of the Calvin cycle, which convert CO₂ into sugar.
  • The Calvin cycle is named after a professor at UC Berkeley.
  • Possibly followed by fatty acid biosynthesis as the very last topic, time permitting.

14. Review: Weak Acids, Bases, and Buffers

• An effective buffer requires:
  1. High molarity — one or two molecules will not control pH; research buffers are typically 50 mM or higher.
  2. pKa near the desired pH — choose a compound whose pKa is close to the target pH (the Sagal textbook appendix lists compounds by pKa).
  3. Appropriate base-to-acid ratio — if the reaction produces protons, the buffer needs more base form (B) than acid form (A), because only B can absorb an incoming proton.
  4. Non-interfering — the buffer should not activate or inhibit the enzyme under study, nor absorb light at wavelengths used for detection.
• Henderson-Hasselbalch equation: pH = pKa + log([B]/[A]).
  • At pH = pKa, the buffer is 50% base and 50% acid.
  • When pH is well above the pKa, the compound is mostly in the base form.
• When protons are added: the new [B] decreases by the amount of protons added; the new [A] increases by that amount. Too much acid overwhelms the buffer and pH plummets.
• When protons are removed (via membrane transport or a chemical reaction incorporating the proton): pH rises; the buffer's A form donates protons to compensate.

15. The Pyruvate-to-Lactate Reaction and NADH

• Reaction: Pyruvate + NADH + H⁺ --[Lactate dehydrogenase (LDH)]--> L-Lactate + NAD⁺
• This reaction is reversible.
• Three substrates (pyruvate, NADH, H⁺) and two products (L-lactate, NAD⁺).
• NAD = nicotinamide adenine dinucleotide. NADH is the reduced form; NAD⁺ (superscript +1) is the oxidized form.
• Pyruvate structure: three carbons; C1 is a carboxyl group (pKa 2.5). At physiological pH (~7), the carboxyl is fully deprotonated (base form), which is why it is called pyruvate rather than pyruvic acid. Students must learn structures that predominate at pH 7.
• NADH carries a hydride — two electrons plus one proton (H⁻).
• Three oxidation states of hydrogen:
  • H⁺: no electrons.
  • H· (H dot): one electron, no charge.
  • H⁻ (hydride): two electrons, negative charge.

  Reference figure · external source

• NADH is a safe energy carrier: it does not release its energy when it bumps into membranes, proteins, or DNA. Only an enzyme (e.g., LDH) can unlock the energy at its active site.
• NADH is an important player in many pathways, not just glycolysis.
• Lactate stereochemistry: LDH always produces L-lactate (OH on C2 drawn on the left). Enzymes are stereospecific — they do not alternate between L- and D-forms.
• When the reaction proceeds in the pyruvate-to-lactate direction, a proton is consumed (incorporated into lactate), which would raise pH if not for the buffer.
• This is the last step of glycolysis.

Exam emphasis: Glycolysis must be known thoroughly for the first midterm — structures, enzyme names, and whether each reaction is reversible. The full glycolysis pathway will be on the last page of Homework 1.

16. Buffer Preparation Problem: Phosphate Buffer at pH 6.8

• Goal: prepare 1 L of 50 mM phosphate buffer at pH 6.8.
• Available reagents: 1 M phosphate buffer at pH 12, 2 M HCl, 3 M NaOH.
• Step 1 — Determine volume of phosphate stock: 50 mmol needed / 1 M = 50 mL of stock solution. Most of the final volume will be water.
• Polyprotic species: phosphoric acid has three pKa values. Students should write out all species and their pKa values.
• Step 2 — Henderson-Hasselbalch at initial pH 12: pH 12 is near pKa₃ (12.32). Calculate the ratio of PO₄³⁻ (base) to HPO₄²⁻ (acid). Result: 0.4786 B per 1 A, so B = 32% of total phosphate. This is reasonable because pH 12 is below pKa₃ 12.32, so less than 50% is in the fully deprotonated form.
• Step 3 — Henderson-Hasselbalch at final pH 6.8: pH 6.8 is near pKa₂ (7.21). Calculate the ratio; result: A = 72%. This is reasonable because pH 6.8 is below pKa₂ 7.21, so more than 50% is in the acid form.
• Step 4 — Determine acid needed: must convert the 32% in PO₄³⁻ all the way down, then create 72% in the H₂PO₄⁻ form. Total: 1.04 equivalents of protons per mole of phosphate.
• Step 5 — Volume of HCl: 50 mmol phosphate × 1.04 = 52 mmol H⁺. From 2 M HCl: 52 mmol / 2 M = 26 mL.
• NaOH is not used because the pH must come down from 12 to 6.8.
• Step 6 — Add water to bring total volume to 1 L (1000 − 50 − 26 = 924 mL water). Without the water, the molarity would be far higher than 50 mM.
• Shortcut for two-unknown problems: divide the Henderson-Hasselbalch ratio by 1, giving B per (B + A) directly without setting up two simultaneous equations.

17. Man-Made Buffers: Tris

• Phosphate is prevalent in biological molecules (DNA, RNA, phospholipids), so using it as a research buffer can activate or inhibit the enzyme under study.
• Man-made buffer compounds are preferred — substances cells have never encountered.
• Tris structure: three CH₂OH groups (hence "tris") attached to a central carbon, plus one amino group. The CH₂OH groups form hydrogen bonds with water, making tris instantly water-soluble.
• Only one proton (on the amino group) can dissociate, so tris has a single pKa (~8.2) and one inflection point in its titration curve.
• Acid form (protonated amino group): +1 charge.
• Base form (lone pair on nitrogen): 0 charge.
• pKa of ~8.2 falls within the biological pH range, making tris a common choice.
• To prepare a tris buffer: start with tris base, add HCl to bring the pH down into the flat buffering region of the titration curve.

18. Enzyme Kinetics: Michaelis-Menten vs. Allosteric Enzymes

• Michaelis-Menten enzymes:

  • Plot of initial velocity (v₀) versus substrate concentration [S] produces a hyperbola.
  • At zero substrate, velocity is zero; velocity rises steeply at low [S], then plateaus as the enzyme becomes saturated.
  • Equation: v₀ = Vmax·[S] / (Km + [S]).
  • Most enzymes in glycolysis (including LDH) are Michaelis-Menten enzymes.

• Allosteric enzymes:

  • Velocity-versus-substrate plot produces a sigmoidal (S-shaped) curve.
  • Velocity starts slowly at low [S] but then rises very steeply.
  • Equation: similar to Michaelis-Menten but with [S] raised to the nth power — v₀ = Vmax·[S]ⁿ / (K₀.₅ⁿ + [S]ⁿ).
  • n (cooperativity coefficient): measures cooperativity of subunits. Allosteric enzymes almost always have subunits that communicate via weak bonds. A larger n (e.g., 2.3) means the curve rises more steeply than a smaller n (e.g., 1.8).
  • Over the same range of substrate concentrations, an allosteric enzyme shows much greater change in velocity than a Michaelis-Menten enzyme — allosteric enzymes are more sensitive to changes in substrate levels.
  • An allosteric inhibitor shifts the sigmoidal curve to the right (degree depends on inhibitor concentration).
  • An allosteric activator shifts the curve to the left.
  • A couple of enzymes in glycolysis are allosteric.

• Metabolic control:

  • Cells have diverse metabolic demands; enzymes in a pathway cannot run unchecked.
  • Example: glucose 6-phosphate (product of the first step of glycolysis) does not always continue down glycolysis — it sometimes enters the pentose phosphate pathway. Allosteric enzymes act as valves controlling the flow of metabolites between pathways.
  • Reactions with large negative ΔG values must be tightly controlled; these are usually catalyzed by allosteric enzymes, though other regulatory mechanisms exist.

19. Affinity Chromatography Introduction

• Purpose: purify a specific biomolecule from the complex mixture obtained by breaking open cells.
• The technique exploits biological specificity — the target enzyme does something unique that distinguishes it from all other proteins, even those with similar molecular weight or charge.
• The column contains microscopic beads (the matrix): flexible, made from a cross-linked polymer of sugars, with pores that buffer and molecules can enter.
• A ligand is covalently attached to the bead to capture the target protein.
• The bead must be polar: most surface residues of a folded enzyme are polar; its nonpolar residues are buried in the interior. A nonpolar bead would cause the enzyme to unfold (exposing its nonpolar core to bind the bead), destroying its structure and activity.
• Lecture ended here; affinity chromatography continues in the next session.