BIS 103 -- Midterm 1 Study Guide

Included lectures:

Abbreviations

Prerequisites (BIS 102)

• Amino acid structures and one-letter codes (used without reteaching)
• Four levels of protein structure (primary through quaternary)
• Basic buffer chemistry and titration curves
• Spectrophotometry fundamentals

L1 -- Course Introduction and Metabolism Overview (2026-08-03)

Exam Structure and Grading

• Two midterms (50 min each) + one final (1 hr 50 min; most finish in ~1 hr)
• Final = 200 pts (2x midterm value); half covers post-MT2 material, half covers MT1+MT2
• Grading curve: class average = lowest B-; top ~15% receive some form of A
• Question types: calculation, short answer, some multiple choice (no Scantron); bring a calculator; all equations/constants furnished
• Exams test on lecture content and homework

EXAM: "You're crazy if you don't do the homework." (L1)

• HW1 is the only assignment for the first midterm (L1, reiterated L2)

Course Themes

The Cell as a Chemical Engine

• Open system: nutrients in (glucose, O2), waste out (CO2, H2O, heat/entropy)
• No cell captures all consumed energy; some is always lost as heat and entropy
• Delta G = free energy for work; Delta H = enthalpy; T = temp (K); Delta S = entropy
• Delta G-prime for glucose --> CO2 + H2O = -686 kcal/mol
• Delta G-prime for ATP synthesis (ADP + Pi --> ATP) = +7.2 kcal/mol
• Pi structure at ~pH 7: P double-bonded to one O, two O- groups, one OH
• Course uses kcal/mol (Sagal textbook); multiply by a constant to convert to joules

Three Techniques for Overall Cellular Metabolism (Overview)

• Using multiple techniques provides different perspectives; relying on one method can be misleading (L1)

Mitochondria and Chloroplast Preview

• Mitochondria: PDH complex, Krebs cycle, fatty acid breakdown (matrix); ETC + ATP synthase (inner membrane); bacteria have ATP synthases in their inner membranes
• Chloroplasts: Photosystems 1 & 2 in thylakoid membranes (light reactions); Calvin cycle enzymes in stroma (CO2 --> sugar)

Buffers and Henderson-Hasselbalch

• Effective buffer requires: (1) high molarity (~50 mM+), (2) pKa near target pH, (3) appropriate B:A ratio (more B if reaction produces H+), (4) non-interfering with enzyme/detection
• Henderson-Hasselbalch: pH = pKa + log([B]/[A])
  • At pH = pKa: 50% B, 50% A
  • pH well above pKa: mostly base form
• When H+ added: [B] decreases, [A] increases by amount added; excess acid overwhelms buffer
• When H+ removed (e.g., consumed in a reaction): pH rises; A donates H+ to compensate

Buffer Preparation Problem: Phosphate Buffer at pH 6.8

• Goal: 1 L of 50 mM phosphate buffer at pH 6.8 from 1 M phosphate stock at pH 12, 2 M HCl, 3 M NaOH
• Volume of stock: 50 mmol / 1 M = 50 mL
• H-H at pH 12 (near pKa3 = 12.32): B (PO4 3-) = 32% (pH below pKa3, so < 50% fully deprotonated)
• H-H at pH 6.8 (near pKa2 = 7.21): A (H2PO4-) = 72% (pH below pKa2, so > 50% acid form)
• Total proton equivalents: 1.04 per mol phosphate --> 50 mmol x 1.04 = 52 mmol H+
• Volume HCl: 52 mmol / 2 M = 26 mL (NaOH not used -- pH must come down)
• Water: 1000 - 50 - 26 = 924 mL
• Shortcut: divide H-H ratio by 1, giving B/(B+A) directly without simultaneous equations

Man-Made Buffers: Tris

• Phosphate is in many biomolecules (DNA, RNA, phospholipids) and can interfere with enzymes; man-made buffers avoid this
• Tris structure: three CH2OH groups + one amino group on central carbon; CH2OH groups H-bond with water (instantly soluble)
• Single pKa (~8.2): acid form (protonated NH3+) = +1 charge; base form (lone pair on N) = 0 charge
• Preparation: start with tris base, add HCl to desired pH

Pyruvate-to-Lactate Reaction and NADH

• Pyruvate + NADH + H+ --[LDH]--> L-Lactate + NAD+ (reversible; last step of glycolysis)
• Three substrates, two products
• NADH carries a hydride (H-): two electrons + one proton
• Three oxidation states of H: H+ (no e-), H-dot (one e-), H- (two e-)
• NADH is a safe carrier: does not release energy upon bumping into membranes/proteins/DNA; only unlocked at enzyme active sites
• Pyruvate: 3 carbons; C1 carboxyl pKa 2.5 --> fully deprotonated at pH 7 (hence "pyruvate," not "pyruvic acid")
• LDH always produces L-lactate (OH on C2 drawn left); enzymes are stereospecific
• When reaction proceeds pyruvate --> lactate, a proton is consumed, which would raise pH without buffer

EXAM: Glycolysis must be known thoroughly for the first midterm -- structures, enzyme names, and whether each reaction is reversible. (L1)

Enzyme Kinetics: Michaelis-Menten vs. Allosteric

• Kinetics = how fast; thermodynamics = potential. A reaction can have large negative Delta G but without an enzyme has no chance kinetically.

• Larger n --> steeper sigmoidal curve
• Reactions with large negative Delta G need tight control; usually catalyzed by allosteric enzymes
• Most glycolytic enzymes are Michaelis-Menten; a couple are allosteric (L1); specifically HK, PFK1, PK (L4)
• G6P can enter pentose phosphate pathway instead of continuing glycolysis; allosteric enzymes control which route (L1)

Affinity Chromatography (Introduction)

• Purpose: purify a specific biomolecule using biological specificity
• Column beads: microscopic, polar, flexible, cross-linked sugar polymer, porous
• Bead must be polar because most surface residues of a folded enzyme are polar; a nonpolar bead would unfold the enzyme
• See L2 for full affinity chromatography coverage

Key Definitions (L1)

L2 -- DNA Microarrays, 2D Gel Electrophoresis, and Lipid Review (2026-08-04)

Affinity Chromatography (Full Treatment)

• Beads are porous; the column is not performing molecular sieving or separating by size
• A covalent arm (~6 carbons recommended) lifts the ligand from the bead surface
  • Too short (1-2 C): arm does not function
  • Too long (12+ C): arm acts as hydrophobic coating
• Ligand: covalently attached to arm; can be substrate, product, inhibitor, coenzyme, or cofactor (metal ion)
• For enzymes with two substrates (e.g., HK uses glucose + ATP): only one substrate on the column; providing both would allow catalysis
• Enzyme binds ligand at active site via weak bonds (ionic, H-bond, van der Waals, hydrophobic effect); binding is dynamic but many ligands per bead retain the enzyme
• All other molecules flow through

Analytical Gel Electrophoresis

• Stacking gel (~4%): compresses sample into thin band before entering resolving gel (~10%); prevents band spreading from diffusion (entropy)
  • Wells formed by Teflon comb (~15 uL capacity each)
  • Stacking requires different pH values in stacking gel, resolving gel, and upper buffer (resolving gel pH 8.8, stacking gel pH 6.8)
  • IEF gels do not need stacking because proteins focus to sharp bands at PI as long as power is applied
• Bromophenol blue: tracking dye; does not interact with proteins; runs faster than any protein; signals when to stop
• SDS: 12-carbon man-made strong detergent (full -1 charge)
  • Strong detergent = full charge; mild detergent = polar groups only
  • Hydrophobic tail binds hydrophobic R groups (Leu, Ile, Trp, etc.); does not bind charged/polar residues (Glu, Arg) -- leaves gaps
  • Larger subunit binds proportionally more SDS; thousands of negative charges overwhelm native charge --> same Q/M for all proteins
  • SDS-coated denatured subunits repel each other --> clustered micelles
  • Mercaptoethanol added in excess to reduce disulfide bonds (atmospheric O2 can create artificial disulfides)

EXAM: Correct electrode placement for SDS-PAGE is a likely MT1 question. SDS-coated proteins are heavily negative --> positive electrode at bottom. Negative electrode at bottom would drive proteins up into buffer. (L2)

Fatty Acids

• Carbon 1 = carboxyl carbon; first number = total carbons; after colon = number of double bonds; parenthetical = positions
• All double bonds are cis (enzymes are stereospecific); cis creates a kink affecting membrane fluidity
• Ionized (COO-) form changes the name (e.g., palmitic acid --> palmitate)
• Free fatty acids act as detergents (compare SDS: 12C + charge vs. palmitate: 16C + charge); only sick/dying cells have lots of free fatty acids; normally covalently attached
• Stearic acid (18C) contains far more energy per molecule than glucose (6C)

EXAM: Students are typically asked to number the carbons in fatty acid structures. (L2)

Lipids: Acylglycerols

• Glycerol backbone with fatty acids at C1, C2, C3 via ester bonds
• Triacylglycerol (three fatty acids) = fat; energy deposit, not in membranes
• L-glycerol: OH on C2 drawn left; most organisms use L-form; archaea use D-glycerol
• Unique enzyme for each glycerol position (attach and remove); enzymes distinguish L- from D- using ~20 weak bonds

Lipids: Phospholipids

• Membrane construction (plasma membrane and organelle membranes)
• Structure: glycerol + fatty acid at C1 + fatty acid at C2 + phosphate at C3 linked to an alcohol via phosphodiester
• Phosphodiesters also in DNA/RNA backbones

• Phosphate is always at C3; the enzyme is stereospecific and never makes a mistake (mutation would likely be lethal)

EXAM: Students may be asked to draw a specific phospholipid at a given pH with designated fatty acids at each position. (L2)

DNA Microarrays (Full Procedure)

mRNA isolation:

• Extract all RNA with organic solvent; only mRNA is wanted
• mRNA distinguished by poly-A tail at 3' end (tRNA, rRNA lack it)
• Affinity column with oligo-dT ligand (covalently attached directly to bead; no arm needed -- mRNA is linear)
• mRNA poly-A tails bind oligo-dT via H-bonding (A-T); dynamic binding; abundance of oligo-dT retains mRNA
• tRNA and rRNA flow through
• Two identical columns: one for cancer cells, one for normal cells (control)
• Fritted disc at bottom traps beads
• Nucleic acids absorb UV at 260 nm (A260; conjugated double bonds in bases)
• Flow-through: high A260 fractions = tRNA + rRNA (discard); wash with buffer (~pH 7.5) until A260 = 0

Elution:

• Change pH by large amount (disrupts H-bonds) or decrease salt concentration
• Decreasing salt removes cation shielding from phosphate backbone negative charges --> strands repel and separate

cDNA synthesis and labeling:

1. Add excess oligo-dT primers (anneal to poly-A tail for double-stranded starting point)
2. Add reverse transcriptase
3. Add dNTPs (dATP, dGTP, dCTP, dTTP) -- energy sources and building blocks
4. One dNTP (e.g., dATP) made fluorescent (emits at longer wavelength when excited)
5. Reverse transcriptase synthesizes cDNA from mRNA template

• Cancer cell mRNA --> red fluorescent cDNA; normal cell mRNA --> green fluorescent cDNA
• Two outcomes: mRNA gains visible color; copy is DNA (more stable than RNA)

Removing mRNA template:

• Incubate mRNA-cDNA duplex in 1 M NaOH + heat for 15 min
• Mechanism: OH- deprotonates 2'-OH of RNA sugar --> 2'-O- attacks partial-positive phosphorus --> breaks phosphodiester bond at every nucleotide --> fragments mRNA completely
• DNA is unaffected (no 2'-OH; has only H at 2' position)
• Critical: add HCl to neutralize back to ~pH 7 (cDNA cannot H-bond to chip at high pH)

The chip:

• Microscope slide; each dot = multiple copies of one gene's ssDNA (~1 nL volumes, machine-deposited)
• Adjacent dots = different genes; each dot must have ~same copy number
• ssDNAs act as receptors; gene identity at each position known only to manufacturer

Hybridization and scanning:

• Equal amounts red + green cDNA incubated with chip + buffer (optimized); rotated
• Rinse to remove non-specific binding; place in fluorescence scanner

• Scanner images red and green channels separately, then merges
• Red spots identify genes with altered expression in cancer; not all may be direct causes (one upstream gene could control many targets)

2D IEF-SDS PAGE (Full Procedure)

Advantage over microarrays: A single base substitution changing a residue's charge (e.g., Val --> Glu) shifts the protein on IEF but may not be detected by a microarray (rest of mRNA still binds). SDS-PAGE alone will not detect charge changes.

First dimension -- IEF tube gel:

• Glass tube, gel polymerizes in ~15 min
• Components: acrylamide (monomer), bisacrylamide (cross-linker; without it no gel forms), ampholytes (small molecules creating pH gradient when current applied), TEMED (prolongs free radical lifetime), ammonium persulfate (persulfate bond breaks in water --> sulfate free radicals --> initiate polymerization chain reaction)
• Electrodes: top = 60 mM NaOH (high pH), bottom = 60 mM H2SO4 (low pH); extreme pH traps ampholytes in gel
• Broad pH gradient (pH 2-10) for all proteins; sample loaded on top; proteins migrate to PI and stop
• Hundreds of bands possible; some proteins may co-migrate

SDS treatment of extruded IEF gel:

• Gel extruded from tube with plunger (like spaghetti) into SDS solution
• SDS diffuses into gel; proteins too large to diffuse out
• SDS + mercaptoethanol + heat: separates subunits, completely unfolds (only primary structure remains)

Second dimension -- SDS-PAGE slab gel:

• Single large well (Teflon comb) to accommodate entire IEF gel
• Pour resolving gel first, then stacking gel; place IEF gel in well
• Stacking ~4%, resolving ~10%; SDS throughout
• Positive electrode at bottom (SDS-coated proteins heavily negative, migrate down)
• Bromophenol blue + glycerol on top for tracking; stop when dye nears bottom
• Result: 2D map; vertical line = same PI; horizontal line = same MW; smaller proteins toward bottom
• Stain to visualize; merge cancer vs. normal images to find differences
• Sensitivity enhancement: grow cells on S-35 methionine + autoradiography (~1 week in freezer)

L3 -- Microcalorimetry and Introduction to Glycolysis (2026-08-05)

Microcalorimetry (Full Treatment)

• Third technique for examining cells; measures very small heat flows (microwatt range) from living cells
• Differs from freshman chem calorimetry: cells stay alive, not burned to completion
• Can also measure CO2 production rate and O2 consumption rate
• These three measurements cannot be obtained from microarrays or 2D gels (L3)
• Delta G = Delta H - T Delta S; microcalorimeter measures only Delta H

EXAM: The Gibbs free energy equation is always provided on exams. (L3)

Instrument setup:

• Ampule: small metal container (~1 mL), special heat-conducting alloy; welded metal cylinder inside creates an upper well (~40 uL)
• Tissue (leaves, bacteria, RBCs, etc.) in main chamber; must not contact liquid in upper well
• Metal lid screws on; small hole fitted with thin tubing to pressure sensor (thin to prevent external heat conduction)
• Instrument: 4 openings -- 1 reference (empty, never changed), 3 sample positions; each surrounded in 3D by heat sensors --> electrical signals --> computer (separate channels)
• Large lid covers entire instrument to block room heat

Part A -- Baseline heat rate (~30 min):

• Upper well: 40 uL water
• y-axis: microwatts/mg dry weight; x-axis: time (min)
• Initial signal off-scale (room heat); recovers and levels off at plateau = heat flow from living cells
• If signal = 0, cells are dead

Part B -- CO2 trap (~30 min):

• Replace water with 40 uL of 0.4 M NaOH (strong base, deliberately in excess); NaOH never touches tissue
• Signal levels off at higher plateau than Part A
• Difference (B - A) is proportional to CO2 production rate
• Mechanism: OH- attacks partial-positive C of CO2 --> bicarbonate; excess OH- converts to carbonate; reactions are exothermic
• CO2 is nonpolar, crosses membranes freely; in sealed ampule, bounces at high velocity (ideal gas); hitting NaOH surface = chemically trapped as carbonate + heat
• NaOH in excess ensures every CO2 captured
• If Part B = Part A, no detectable CO2 (nonfunctional mitochondria in plant cells)

Part C -- Control (~30 min):

• Replace NaOH with 40 uL water
• Signal should return to ~Part A baseline
• Verifies Part B caused no tissue damage (e.g., NaOH dripping onto tissue)

Why water in Part A: Controls for water vapor; NaOH solution is mostly water. Ensures Part B effect is due to NaOH, not water vapor changing cell metabolism.

Dry weight determination: After experiment (~90 min total), tissue dried in vacuum oven at 90 C overnight (vacuum prevents oxidation); weighed on precise balance.

Pressure sensor and O2 measurement:

• Pressure data collected simultaneously throughout
• Parts A and C: multiple gases (N2, O2, CO2) --> cannot separate individual contributions
• Part B: NaOH eliminates CO2, N2 is inert --> any pressure decrease = O2 consumption (mainly mitochondria)
• Calculate picomoles O2/min using PV = nRT

Summary of output: Heat rate/mg dry weight, CO2 production rate, O2 consumption rate (all with real numerical values)

Application: Predicts tree growth potential in 2-3 days from small tissue samples (vs. years of field observation); also applicable to cancer cells; many replicates (~20) for averaging

Intermediary Metabolism

• Studies small organic compounds (~1,000 MW or less): ATP, glucose, fatty acids, amino acids -- not macromolecules
• Catabolic reactions break down compounds; converge on acetyl-CoA
• Anabolic reactions build compounds; diverge/branch from common precursors
• Energy carriers connecting the two: NAD/NADH, NADP/NADPH, FAD/FADH2, ATP
• These exist in small pool sizes; cells do not store large quantities
• To build something (needs ATP, etc.), cell must simultaneously break something down; pathways are perfectly meshed

Pathway types:

• Acetyl-CoA: 2-carbon acetyl group carried by CoA via thioester (S-C=O)

EXAM: Students must know glycolysis and the Krebs cycle thoroughly. (L3)

Coupled Enzyme Reactions: Glutamine Synthetase

• NH4+ at high concentration is toxic; many N-containing compounds break down to ammonia --> ammonium at cell pH

• Glutamate + NH4+ --> glutamine is endergonic alone

• Cells couple it with exergonic ATP breakdown at one enzyme active site:

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

• Mechanism:

  1. ATP phosphorylates side-chain carboxyl of glutamate (never the alpha-carboxyl; enzyme orients correctly) --> acyl phosphate intermediate + ADP
  2. NH4+ enters active site; active-site group removes H+ --> NH3; nitrogen lone pair (nucleophile) attacks electrophilic carbonyl C of acyl phosphate --> displaces phosphate --> glutamine + Pi

EXAM: Acyl phosphates are high-energy compounds (large negative Delta G); recognize them immediately. (L3)

• Coupled reactions were discovered early through glycolysis study; led to incorrect assumption that all energy-transfer reactions use substrate-level coupling (proton gradients discovered later)
• Coupled reactions need not involve ATP

Glycolysis Steps 1-4

Glycolysis = lysis of sugars; occurs in cytosol; glucose --> pyruvate/lactate. Continues in L4.

Glucose structure:

• Beta-D-glucose: 6 carbons; C1 at anomeric position
• Shorthand: vertical lines = OH groups; C2 OH below, C3 OH above, C4 OH below
• C1 OH above = beta (slightly more stable in solution); below = alpha; ring opens/closes (mutarotation)

Glucose transport:

• Glucose is very polar (multiple O atoms); cannot cross nonpolar membrane interior without help
• Glucose transporter: 45,000 MW membrane protein; facilitates diffusion both directions
• Humans have 14 forms (GLUT1-14); brain runs mostly on glucose

Step 1: Hexokinase

Glucose + ATP --[HK; Mg2+]--> G6P + ADP

• Phosphorylates C6 OH specifically; "hexokinase" implies it handles other hexoses too
• At physiological pH, phosphate on C6 carries -2 charge
• Consumes 1 ATP (even though glycolysis goal is to produce ATP)
• Accomplishments: (1) pulls equilibrium (glucose disappears, more enters cell), (2) traps glucose inside (transporter rejects G6P), (3) charged handle on C6 helps next enzyme orient substrate, (4) raises glucose energy slightly
• Mg2+ required: sits near ATP phosphates, shields negative charges so C6 OH (nucleophile, partial negative) can approach and attack phosphorus (partial positive). Without Mg2+, no attack.
• HK conformational change: two domains; open without glucose; glucose binding closes domains like a clamshell, burying glucose and physically excluding water. C6 OH attack is 40,000x faster than water attack. (L3)

Step 2: Phosphoglucose Isomerase (PGI)

G6P --[PGI]--> F6P (reversible)

• Moves bond from ring O to C2; converts C1 from aldehyde to alcohol (aldose --> ketose)
• Purpose: aldehyde C1 is electrophilic (not a nucleophile); converting to OH allows C1 to attack ATP in step 3

Step 3: Phosphofructokinase-1 (PFK1)

F6P + ATP --[PFK1]--> F1,6BP + ADP (one-way, irreversible)

• Phosphorylates C1 OH using a second ATP; glycolysis has now consumed 2 ATPs
• A second isomer (PFK2) exists; discussed later in course

Step 4: Aldolase

F1,6BP --[Aldolase]--> DAP + G3P (reversible)

• Splits F1,6BP between C3 and C4 --> two 3C molecules, each with a phosphate
• Reverse = aldol condensation (L4)
• Cell strategy: phosphorylate C6, phosphorylate C1, split down the middle

L4 -- Glycolysis Completion, Energetics, and Beer's Law (2026-08-10)

Covers sections 1-21 of the source note only. The lecturer stated in section 22: "Material from this point forward is on the second midterm." Post-boundary content (starch, glycogen) is outside this guide's scope.

Glycolysis Steps 5-10

Step 5: Triosephosphate Isomerase (TPI)

DAP --[TPI]--> G3P

• Converts DAP to G3P; glucose effectively yields 2 G3P molecules
• TPI is catalytically perfect: cannot evolve faster; only rate limitation is substrate diffusion
• Without TPI, half of glucose's carbons would be lost

Step 6: Glyceraldehyde-3-Phosphate Dehydrogenase (G3PDH)

G3P + Pi + NAD+ --[G3PDH]--> 1,3-BPG + NADH + H+ (x2 per glucose)

• Dehydrogenases transfer electrons onto NAD, FMN, or FAD; kinases transfer phosphate
• 1,3-BPG contains a high-energy acyl phosphate bond (Delta G-prime ~ -12 kcal/mol)
• If water were used instead of Pi, energy would be lost as heat/entropy; Pi traps energy in acyl phosphate for ATP synthesis in next step
• H+ released comes from Pi (has OH at physiological pH)
• NAD+ reduced by hydride (H-): H and its two bonding electrons transfer from substrate C to NAD
• 1,3-BPG is the cousin of 2,3-BPG (important for hemoglobin)

Step 7: Phosphoglycerate Kinase

1,3-BPG + ADP --[Phosphoglycerate kinase]--> 3-PGA + ATP (reversible; x2 per glucose)

• 3-PGA: C1 = carboxyl group, phosphate on C3
• Net ATP at this point from 1 glucose: 2 consumed (HK + PFK1), 2 produced here --> net = 0 (break even)

Step 8: Phosphoglycerate Mutase

3-PGA --[Phosphoglycerate mutase]--> 2-PGA

• 3-PGA and 2-PGA are structural isomers
• Shell game mechanism: active site already holds a phosphate; places it on C2 before removing the one on C3 (the phosphate on C2 is not the same one that was on C3)
• Direct removal + reattachment would cost too much energy

Step 9: Enolase

2-PGA --[Enolase; removes H2O]--> PEP

• Removes OH on C3 + H on C2 as water --> double bond with phosphate on C2
• PEP Delta G-prime = -13.8 kcal/mol (more than enough to make an ATP)
• PEP's instability: without the phosphate, molecule would tautomerize enol --> keto (more stable); phosphate pins it, preventing tautomerization until next enzyme

Step 10: Pyruvate Kinase

PEP + ADP --[PK]--> Pyruvate + ATP (one-way; x2 per glucose)

• Phosphate released and donated to ADP; keto-enol tautomerization proceeds --> large negative Delta G

LDH and NAD Regeneration

Pyruvate + NADH + H+ --[LDH]--> Lactate + NAD+

• Purpose: regenerate NAD+ (pool size is very small); without this step, G3PDH reaction stalls and glycolysis stops
• Net yield from 1 glucose: 2 ATP

EXAM: Students need to know the steps of glycolysis thoroughly. (L4)

Complete Glycolysis Summary Table

Reference figure · external source

Net: 2 ATP per glucose (both glycolysis and alcoholic fermentation)

Alcoholic Fermentation in Yeast

• Glucose processed through glycolysis to pyruvate; last two steps differ from lactate pathway:

1. Pyruvate --[Pyruvate decarboxylase; Mg2+ (cofactor), TPP (coenzyme)]--> Acetaldehyde + CO2 (one-way)
   • CO2 is nonpolar, leaves the cell
   • Pyruvate decarboxylase is allosteric (one-way reaction)
2. Acetaldehyde --[Alcohol dehydrogenase]--> Ethanol + NAD+
   • Purpose: regenerate NAD+ (same logic as LDH -- small pool size)

• Net: 2 ATP per glucose

EXAM: If asked to draw all steps of alcoholic fermentation, include the entire glycolysis pathway, not just the last two steps. (L4)

NAD Structure and Spectral Properties

Reference figure · external source

• NAD = nicotinamide adenine dinucleotide
• Lower portion = AMP (acts as handle for enzyme orientation; AMP handles also in CoA, FAD, etc.)
• Business end = nicotinamide ring (amide: C=O bonded to NH2, not amine)
• Hydride (H-) placed specifically at top of nicotinamide ring and nowhere else
• Positive charge on N of NAD+ partially delocalized by resonance to C at top of ring --> attracts hydride
• Reduction of NAD+ to NADH eliminates the positive charge on N
• Students should be able to draw both oxidized (NAD+) and reduced (NADH) forms

Allosteric Regulation of Glycolysis

• Generalization: reactions with large negative Delta G --> typically catalyzed by allosteric enzymes (multiple subunits, sigmoidal kinetics)
• Allosteric glycolytic enzymes: HK, PFK1, PK (all catalyze one-way reactions)
• In alcoholic fermentation: pyruvate decarboxylase is also allosteric (one-way)
• Allosteric enzymes are much more sensitive to changes in compound concentrations
• PFK1 regulation:
  • ADP = positive allosteric effector (activator): ADP buildup --> cell low on energy --> PFK1 speeds up
  • ATP = negative allosteric effector (inhibitor): ATP buildup --> cell has enough energy --> PFK1 slows down
  • ADP binds at a site separate from active site, changes conformation, increases rate
• Nothing in metabolism ever goes to zero; negative regulation = rate slows down

Purposes of Glycolysis

1. Produce some ATP (net 2 per glucose)
2. Provide intermediates siphoned off for biosynthesis:
   • 3-PGA --> serine (S) --> glycine or cysteine
   • DAP --> glycerol (backbone of phospholipids and triacylglycerols)
   • 1,3-BPG --> 2,3-BPG in red blood cells (for hemoglobin function; RBCs sacrifice glycolysis to make it)

Equilibrium and Free Energy

• K_eq = [products] / [substrates] at equilibrium
• Cells never reach equilibrium; operate in steady state (open system). Biochemistry uses equilibrium math as a simplification.
• K_eq-prime: K_eq measured at pH 7 (for reactions involving H+)
• Delta G = RT ln([products]/[substrates]) - RT ln(K_eq)
• At equilibrium: Delta G = 0; reaction cannot do work
• If all species at 1 M: ln(products/substrates) = ln(1) = 0 --> chosen as reference to simplify math
  • Delta G-naught = -RT ln(K_eq)
• If all species at 1 M and pH 7: Delta G-prime
• Actual cell free energy: Delta G = Delta G-prime + RT ln(actual product/substrate ratio)
• Delta G-prime values are reference values for comparing reactions in tables; real Delta G depends on actual concentrations (change throughout the day)
• Reversing a reaction: sign of Delta G reverses, K_eq inverts
• Adding (coupling) two reactions: Delta G values add, K_eq values multiply

EXAM: Equations will be provided; students need to understand what they mean. (L4)

Beer's Law

• A = epsilon x L x C
  • A = absorbance (unitless); spectrophotometer measures I0 and I, calculates log10(I0/I)
  • epsilon = molar extinction coefficient (M^-1 cm^-1); large number; determined by molecular structure; requires conjugated double bonds
  • L = path length (cm); standard = 1 cm
  • C = concentration (M, moles/liter); typically very small in biochemistry
• Equation of a straight line: A directly proportional to C, passes through zero
• Absorbance proportional to concentration, not mass; spectrophotometer does not integrate over volume
• Linearity must be verified experimentally; curves off at high concentrations
• Practical test: dilute sample in half, check that A is halved
• Alternative form: A = a x L x C, where little a = specific absorbance; used when MW unknown; C in mg/mL or g/100 mL. Cannot calculate IUs with this form (IUs require micromoles/min --> need MW).

LDH Enzyme Assay and IU Calculation

• Reaction: Pyruvate + NADH + H+ --[LDH]--> Lactate + NAD+
• At 340 nm: only NADH absorbs (pyruvate, H+, LDH protein, buffer do not)
• At t=0, A340 is high (NADH present); if LDH present, A340 decreases as NADH consumed; flat line = no LDH
• Use initial linear region (Beer's Law region); slope = initial velocity (v0)
• Epsilon for NADH at 340 nm: 6,220 M^-1 cm^-1 (would be given on exam)

IU calculation steps:

1. Measure Delta A / time from linear region
2. Convert to per-minute change (e.g., 15 s measurement x4)
3. Calculate Delta [NADH] (M/min) using Beer's Law: Delta C = Delta A / (epsilon x L)
4. Multiply by assay volume (L) to convert M --> moles (liters cancel)
5. Convert moles --> micromoles (x 10^6)
6. Result = IUs of active LDH in cuvette

• Example: 0.15 umol NADH consumed/min = 0.15 IU active LDH
• NADH change is negative (consumed), but IUs reported as positive
• Always carry units through calculations

EXAM: HW1 covers the IU calculation for MT1. HW2 and final extend it further (e.g., IUs in original extract). (L4)

Known-Gaps Appendix

• Post-boundary L4 content (second-midterm material): The lecturer stated in the Aug 10 lecture that material from starch/glycogen onward is on the second midterm. The following topics from the L4 source note are therefore outside this guide's scope: starch structure (amylose, amylopectin), alpha-1,4 and alpha-1,6 glycosidic bonds, reducing vs. non-reducing ends, glycogen structure and function, glycogenin, glycogen synthase, and UDP-glucose. These will be covered in a Midterm 2 study guide.