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Enzymes: Kinetics, Inhibition & the ETC

When a patient on methotrexate gets sick, the enzyme it blocks tells you exactly why: competitive inhibitors mimic substrate, noncompetitive ones don't care how much substrate you throw at them.

BIOCHEMISTRY

Enzymes

Kinetics, inhibition, and the electron transport chain → the machinery that keeps you alive

A patient overdoses on a drug. You flood the system with more substrate and the drug's effect reverses. What type of inhibition was the drug using?
Non-competitive
Competitive
Uncompetitive
Allosteric

What Enzymes Actually Do

Four things. That's it.

Enzymes are catalysts → they make reactions happen faster by lowering the activation energy. They're not consumed. They walk away untouched.
1
Bring substrates together

Think of it like a dating app. Two molecules need to meet, but the body is huge and they'd never find each other on their own. The enzyme grabs both and holds them together → in space and time.

2
Lower the activation energy

Picture a hill. The substrate needs to climb over it to become a product. Without the enzyme, it's Everest. With the enzyme, it's a speed bump. The reaction is now probable, not just possible.

3
Stabilize the high-energy intermediate

At the top of that hill, the substrate is in a high-energy intermediateA fleeting, unstable form of the substrate at the peak of the reaction. If it's not stabilized, it flies apart instead of becoming product. state → like balancing a ball on a peak. Without help, it falls backward. The enzyme holds it steady until the reaction slides down to product.

4
Make reactions go faster

90% of reactions in your body probably wouldn't happen without enzymes. Sure, they're technically possible → but you'd be waiting a thousand years. Enzymes turn "maybe someday" into "right now."

Every Enzyme Has Two Binding Sites

Where things bind determines what happens

ENZYME ACTIVE SITE Substrate (or competitive inhibitor) ALLO- STERIC Non-comp inhibitor fits the keyhole changes shape COMPETITIVE Fights substrate for active site Km ↑ · Vmax same · Overcome with more substrate NON-COMPETITIVE Binds allosteric site, warps enzyme Km same · Vmax ↓ · Cannot overcome
🎯 Active site = where the substrate binds, where the work happens, where competitive inhibition occurs
🎛️ Regulatory site (allosteric site) = the dimmer switch. Speeds up or slows down the enzyme. Where non-competitive inhibition occurs

Competitive vs Non-Competitive

This is THE board question. Know it cold.

Competitive Non-Competitive
WHERE Active site (same as substrate) Regulatory / allosteric site
LOOKS LIKE SUBSTRATE? Yes → that's why it fits No → different shape entirely
Km ↑ increases (less affinity) No change
Vmax No change ↓ decreases
OVERCOME IT? Yes → add more substrate No → you're stuck
% OF DRUGS ~90% (reversible = safe) Reserved for deadly diseases
DRUG ANALOGY Km = 1/Potency, Vmax = Efficacy Km = same, Vmax ↓ = less efficacy
🔑Competitive = Can be overcome. Add more substrate and you win. That's why 90% of drugs are competitive → reversible.
⚠️
Board Trap: "Km increases" doesn't mean more affinity
Km and affinity have an inverse relationship. ↑ Km = ↓ affinity. Higher Km means you need MORE substrate to reach half-max velocity → the enzyme is less interested. Think of Km like "how hard is it to attract the enzyme" → higher = harder.
[Substrate] Velocity Vmax Vmax' Km Km'
Normal
Competitive (↑Km, same Vmax)
Non-competitive (same Km, ↓Vmax)
💊 Why are 90% of drugs competitive? Because they're reversible. If a patient overdoses, flood the system with more substrate and the drug gets outcompeted. Non-competitive drugs are only used when the disease is worse than the side effects.

Delta G → Is This Reaction Gonna Happen?

Negative = yes. Positive = no. That's 80% of it.

📐 ΔG = ΔH − TΔS
ΔG negative → spontaneous, favorable, energy left over
ΔH = heat (enthalpy) • T = temperature • ΔS = entropy (randomness)

The chain: Higher temperature → larger TΔS → more negative ΔG → reaction more favorable. That's why fever speeds up your metabolism. But push past 42°C (107°F) and enzymes denature → game over.

ΔG is additive. A bad reaction can be dragged forward by coupling it with a very favorable one (like ATP hydrolysis). This is how the body runs thermodynamically unfavorable reactions.

Most stable bonds: Carbon-carbon bonds (that's why fat stores so much energy → lots of C-C bonds to break).

⚠️
Board Trap: "Possible" vs "Probable"
Any reaction is possible. The question is whether it's probable. Enzymes don't make impossible reactions happen → they make improbable reactions happen fast. If a question asks "can this reaction occur without an enzyme?" → the answer is yes. It would just take centuries.

OIL RIG

Oxidation Is Loss, Reduction Is Gain → of electrons

Reducing Agent Oxidizing Agent
ΔE Negative (has electrons to give) Positive (wants electrons)
WHAT IT DOES Gives away electrons Accepts electrons
WHAT HAPPENS TO IT Gets oxidized Gets reduced
🔑OIL RIG: Oxidation Is Loss (of electrons), Reduction Is Gain. The reducing agent gets oxidized → it sacrifices itself. The name describes what it does TO others, not what happens to it.

Where 90% of Your ATP Gets Made

Tap each complex to see what it does

I
NADH dehydrogenase
CoQ
carrier
II
succinate dehydrogenase
III
cytochrome bc1
Cyt C
carrier
IV
cytochrome oxidase
V
ATP synthase

Complex I → NADH Dehydrogenase

  • NADH drops off electrons here → regenerates NAD+
  • Pumps H⁺ into the intermembranous space
  • Electrons then ride Coenzyme Q to Complex III

Inhibitors: Amytal, Rotenone

Rotenone is a pesticide that kills bugs by shutting down their Complex I. Amytal is a barbiturate. Both block NADH from donating electrons, so the whole chain stalls.

Coenzyme Q (Ubiquinone)

  • Electron carrier floating in the inner membrane lipid bilayer
  • Shuttles electrons from Complex I/II → Complex III
  • Also called CoQ10 (yes, the supplement)
  • Destroyed by statins → that's one reason statins cause myopathy

Complex II → Succinate Dehydrogenase

  • Double agent: it's BOTH a Krebs cycle enzyme AND part of the ETC
  • Converts succinate → fumarate, producing FADH2
  • Does NOT pump H⁺ → that's why FADH2 = 2 ATP (actually 1.5)
  • Sends electrons to CoQ, then same path as Complex I electrons

Inhibitor: Malonate

Malonate looks like succinate (it's a structural analog), so it competes for the active site. Classic competitive inhibition at Complex II. Board question: "What type of inhibition does malonate cause?" → competitive, because adding more succinate overcomes it.

Complex III → Cytochrome bc1

  • Receives electrons from CoQ
  • Uses heme as part of its structure
  • Pumps H⁺ into intermembranous space
  • Passes electrons to Cytochrome C

Inhibitor: Antimycin

Antimycin is a fish poison (piscicide) used in lake management. Blocks electron transfer inside Complex III. Not commonly tested by name, but shows up as a distractor.

Cytochrome C

  • Second electron carrier, floats in the inner membrane
  • Shuttles electrons from Complex III → Complex IV
  • Also involved in apoptosis (when released into cytoplasm = cell death signal)

Complex IV → Cytochrome Oxidase

  • Where oxygen is finally used → attaches 4 electrons to O₂ → 2 H₂O
  • Contains heme and copper
  • Pumps H⁺ into intermembranous space
  • This is why you need to breathe → O₂ is the final electron acceptor

Inhibitors: CO, Cyanide, Chloramphenicol

CO binds heme iron 200x stronger than O2 (competitive at Complex IV). SpO2 looks normal (pulse ox can't tell the difference). Treat with 100% O2. Cyanide also binds Complex IV iron. Cherry-red skin. Treat with hydroxocobalamin or nitrites+thiosulfate. Chloramphenicol is an antibiotic that inhibits mitochondrial ribosomes (related to Complex IV function). Causes aplastic anemia + gray baby syndrome.

Complex V → ATP Synthase

  • Not really electron transport → this is where phosphorylation happens
  • H⁺ flows back through Complex V (like a turbine in a dam)
  • The flow of protons spins the enzyme and generates ATP
  • This is the chemiosmotic theoryPeter Mitchell's Nobel Prize idea: the proton gradient across the inner membrane is the energy source that drives ATP synthesis. Protons flow down their concentration gradient through Complex V, and that flow powers ATP production.

Inhibitor: Oligomycin

Oligomycin plugs the proton channel in Complex V. H+ can't flow through, so ATP synthase stops spinning. The proton gradient builds up, which ALSO stalls the electron transport chain (because you can't pump more H+ against an already maxed gradient). Result: both ETC and ATP production halt. Distinct from uncouplers, which let H+ leak back WITHOUT making ATP.

🔋 NADH = 3 ATP (actually 2.5) → drops off at Complex I, gets 3 pumping chances
FADH2 = 2 ATP (actually 1.5) → drops off at Complex II, only 2 pumping chances
🔑NADH enters earlier (Complex I), so it gets more pumping stops → more ATP. FADH2 skips Complex I → fewer stops → less ATP. Earlier entry = more reward.

Inhibitors vs Uncouplers

Both kill ATP production. Different mechanisms.

Inhibitors What They Block
Amytal, RotenoneComplex I
MalonateComplex II
AntimycinComplex III
CO, Cyanide, ChloramphenicolComplex IV
OligomycinComplex V (ATP synthase)
🔥 Uncouplers = proton smugglers. They grab H⁺ from the intermembranous space and drag them back into the matrix, bypassing Complex V. Electron transport continues but ATP synthesis stops. All that energy → heat.
Uncoupler Clinical Relevance
DNP (Dinitrophenol)Was used as a weight loss drug (burns fat → heat). Killed people.
Aspirin (high dose)Reye's syndrome → uncouples in liver mitochondria
Free fatty acidsBrown fat in babies → intentional uncoupling to generate heat
ThermogeninThe protein in brown fat that does the uncoupling
⚠️
Board Trap: CO Poisoning Looks Normal
CO binds iron 200x stronger than O₂. It's a competitive inhibitor at Complex IV. Here's the trap: O₂ saturation (SpO₂) appears NORMAL because the pulse ox can't tell CO-bound hemoglobin from O₂-bound hemoglobin. pO₂ (dissolved oxygen) is also normal initially. Treatment: supplemental O₂ (add more substrate to outcompete). The history is the best clue → space heater, garage, wintertime.

Uncouplers cause: Body temp rises → muscles can't relax → malignant hyperthermia and neuroleptic malignant syndrome

Microsteatosis (small fat droplets in liver): Pregnancy, Tylenol poisoning, Reye's syndrome
Macrosteatosis (big fat droplets in liver): Obesity, Alcohol

How to Name Any Enzyme

First name = substrate. Last name = what you did to it. 90% of the time.

📝 Lactate dehydrogenase = Substrate is lactate, and you dehydrogenated it (removed a hydrogen). It's a recipe. Every enzyme name tells you what happened.

TAP TO REVEAL what each enzyme type does

Kinase
Phosphorylates using ATP (cofactor: Mg²⁺)
Phosphorylase
Phosphorylates using free Pi (no ATP)
Dehydrogenase
Removes hydrogen, uses a cofactor (NAD⁺, FAD)
Carboxylase
Uses CO₂ to make C-C bond (needs ATP + biotin)
Isomerase
Same formula, different structure
Mutase
Moves a side chain between carbons
Transferase
Moves a group from one substrate to another
Lyase
Cuts C-C bonds (no ATP needed → that's Ligases)
Synthase
Stacks substrates together (no ATP)
Synthetase
Stacks substrates together (uses ATP)
Hydrolase
Uses water to break a bond
🔑Synthase vs Synthetase: the extra "t" stands for "takes ATP." Synthetase uses ATP. Synthase doesn't. The longer word = more energy needed.

Elimination Game: Name That Poison

A patient is dying. Clues are coming in. Eliminate suspects until you find the killer.

Oligomycin
Complex V blocker
Cyanide
Complex IV blocker
DNP
Uncoupler
Carbon Monoxide
Complex IV blocker
Loading clue...

Clinical Vignettes

6 patients just walked in. Don't panic → you know this.

Enzyme Classes: Board Style

Two vignettes. One concept each. Tap an answer to check.

Question 1 of 2
A researcher adds a compound that cleaves a peptide bond between two amino acids by incorporating a water molecule into the reaction. No ATP is required and no new chemical group is transferred. Which class of enzyme is responsible?
Kinase
Lyase
Hydrolase
Isomerase
Question 2 of 2
A student is studying four enzymes: trypsin, lipase, alkaline phosphatase, and DNase I. She claims all four belong to the same enzyme class. Which of the following best justifies her claim?
All four are oxidoreductases that transfer electrons
All four are transferases that move phosphate groups
All four are hydrolases that use water to cleave different types of chemical bonds
All four are ligases that form bonds using ATP