Sample note · Biology 101

Enzymes and Enzyme Kinetics

A 38-minute class · 13 sections · 2 charts · about 2,200 words

This is what Nightsesh wrote from one 38-minute biology class, unedited. The class is a sample we wrote for Nightsesh: a professor, lab data on the board, a practice problem and questions from students. While it played, the student tapped Lost me twice and This matters twice and typed two quick jots. Make my notes did the rest in about three minutes. In the app, every ▶ plays the class from that moment.

Enzymes are protein catalysts that speed reactions by lowering activation energy, never by changing ΔG. Using the class's beta-galactosidase lab data, the class built the saturation curve, defined Vmax and Km, and practiced the Michaelis–Menten equation. It then covered temperature and pH optimums, cofactors, competitive vs noncompetitive vs irreversible inhibitors, and allosteric feedback inhibition.

16 stressed✎ 2 your jots13 sections

Your teacher stressed

The five exam points plus two warnings about energy.

Spontaneous does not mean fast. ΔG tells direction, not speed. ▶ 2:00
Know the energy diagram (reactants, hump, transition state, products). It will be on the exam. ▶ 2:30
Enzymes lower activation energy. They never change ΔG or the equilibrium. ▶ 5:30
Know the four ways an enzyme lowers activation energy. ▶ 10:00
Low Km = high affinity. High Km = low affinity. ▶ 17:00
Guaranteed on the exam: use Michaelis–Menten to find a rate from Km and Vmax, and read Vmax and Km off a curve. ▶ 20:00
Temperature and pH have optimums; past the optimum the enzyme denatures. ▶ 37:30
Tell competitive from noncompetitive inhibition from a graph: look at the high-substrate end. ▶ 33:30

Where you got lostAI explained

Nightsesh filled in what your teacher was getting at, using your class and standard background. Check it against the moment.

Why a low Km means tight binding, and how noncompetitive inhibitors work.

? 16:40 · you tapped Lost me

Your teacher was explaining what Km says about binding: a low Km means the enzyme holds its substrate tightly (high affinity). ▶ 16:30
Km is the substrate concentration where the rate reaches half of Vmax.
So Km answers: how much substrate does this enzyme need to get to half speed?
If only a little substrate is needed, the enzyme must be catching substrate easily, so it binds tightly. Small number = high affinity.
If a lot of substrate is needed, the enzyme holds on loosely and lets go often. Big number = low affinity.
e.g. Enzyme A has Km = 0.1 mM; enzyme B has Km = 10 mM. At 0.1 mM substrate, A is already at half speed while B has barely started. A has the higher affinity.
Memory tip Read Km as "how much it takes to get going". Needing less means a stronger grip.

? 32:15 · you tapped Lost me

Your teacher was introducing noncompetitive inhibition: the inhibitor binds somewhere other than the active site and changes the enzyme's shape. ▶ 32:00
The inhibitor attaches to a different spot on the enzyme, not the active site.
That binding changes the enzyme's overall shape, including the active site, so the enzyme does its chemistry poorly or not at all.
Substrate and inhibitor are not fighting over the same spot, so adding more substrate cannot push the inhibitor off.
In effect, some enzymes are taken out of action. The top speed (Vmax) drops, but the enzymes still working bind substrate as usual, so Km stays the same.
e.g. Using the class's checkout-line picture: a noncompetitive inhibitor is like sending 2 of the 5 cashiers home. A longer line of customers can't raise the store's top speed, but each open cashier works just as before.
The fomepizole mention just before this is still competitive inhibition: it blocks alcohol dehydrogenase by competing for the active site, like ethanol.

Free energy and activation energy

ΔG says if a reaction can go; activation energy decides how fast.

ΔG Change in free energy. Negative ΔG = exergonic: releases energy, spontaneous. Positive ΔG = endergonic: needs an energy input. ▶ 0:30
e.g. Oxidizing glucose fully to carbon dioxide and water: ΔG ≈ −2870 kJ/mol. Very spontaneous. ▶ 1:00
Spontaneous does not mean fast. ΔG shows which direction a reaction can go, nothing about its speed. ▶ 2:00
e.g. Sugar in a sugar bowl sits next to oxygen for years without burning: the reaction is downhill, but there's a hill in the way first. ▶ 1:30
Energy diagram: y axis = free energy, x axis = reaction progress. Reactants start high, products end lower (ΔG negative), with a hump between. ▶ 2:30
Transition state The high-energy, unstable arrangement at the top of the hump; bonds are partly broken and partly formed.
Activation energy (Ea) The energy needed to get up to the top of the hump. ▶ 3:00
At room temperature almost no sugar molecules can get over the hump. A flame gives some enough energy; the energy they release pushes more over, so it keeps going. ▶ 3:30
Cells at 37 °C can't use heat like that; they would cook their own proteins.

Enzymes as catalysts

Enzymes lower the hump, not the start or end point.

Enzyme A biological catalyst, almost always a protein. (Ribozymes are RNA catalysts, but in this unit enzyme = protein.) ▶ 4:00
Catalyst Speeds up a reaction without being used up; it comes out unchanged and can work again, thousands to millions of times. ▶ 4:30
An enzyme gives the reaction a different path with a lower hump: lower Ea. Same reactants, same products, same start and end heights.
Lower hump → more molecules make it over at body temperature → faster reaction. ▶ 5:00
Watch out An enzyme does not change ΔG, the energy released, or where equilibrium lies, and cannot make an endergonic reaction exergonic. It only changes Ea, so only how fast you get there. ▶ 5:30
Memory tip Enzymes change Ea, never ΔG.
e.g. Catalase breaks toxic hydrogen peroxide into water and oxygen (the fizz on a cut). One molecule handles about 40 million H₂O₂ per second. ▶ 6:00
e.g. An enzyme in the pathway making DNA building blocks: without it the reaction's half-life is about 78 million years; with it, milliseconds. A speed-up of about 10¹⁷. ▶ 6:30
No need to memorize these numbers; just get a feel for how huge the speed-up is. ▶ 5:30

The active site and specificity

Shape and chemistry of the active site decide what binds; binding reshapes the enzyme.

Active site A pocket or groove on the enzyme's surface where the substrate binds. ▶ 7:00
Substrate The molecule the enzyme acts on. ▶ 7:30
1. Substrate enters the active site. ▶ 8:00
2. The enzyme–substrate complex (ES) forms; the substrate is held by weak bonds (hydrogen, ionic) to amino acids lining the site.
3. The substrate is converted to products while bound.
4. Products are released.
5. The active site is free again and the cycle repeats.
Enzymes are very specific. Lactase breaks down lactose (milk sugar) but not sucrose (table sugar), though both are two-sugar molecules. ▶ 8:30
Specificity comes from the site's shape and the R groups lining it (charged, polar, hydrophobic). Only the right shape and charge pattern binds well. ▶ 9:00
Lock and key Old model: substrate is the key, active site the lock. Not wrong, but too rigid.
Induced fit Better model: as the substrate binds, the enzyme changes shape slightly and clamps around it.
e.g. Hexokinase (first enzyme acting on glucose): when glucose binds, its two halves swing together like a jaw closing. ▶ 9:30
Four ways an enzyme lowers activation energy: ▶ 10:00
1. Orientation: holds two substrates together in exactly the right position to react.
2. Strain: stretches or bends substrate bonds toward the transition-state shape, so they break more easily.
3. Microenvironment: e.g. a pocket more acidic than the rest of the cell, due to the amino acids lining it. ▶ 10:30
4. Direct participation: active-site amino acids briefly form a covalent bond with the substrate, then let go.

Saturation, Vmax and Km

Rate rises with substrate, then levels off when every enzyme is busy.

Lab setup: beta-galactosidase (breaks down lactose) given ONPG, a look-alike; one product is yellow. Yellow was measured with a spectrophotometer at 420 nm and converted to a rate (nmol product/min). Enzyme amount fixed, substrate varied. ▶ 11:30
At low substrate, doubling it nearly doubles the rate (15 → 27 → 41). At high substrate, doubling from 8 to 16 mM only raised it from 70 to 74. ▶ 13:00
Plot: rate on y, substrate concentration on x. It rises steeply, bends over and flattens toward a ceiling it never quite reaches. ▶ 13:30
Saturated Every active site is occupied almost all the time. The bottleneck is no longer finding substrate but how fast each enzyme does the chemistry. ▶ 14:30
e.g. Checkout line with five cashiers: with three shoppers, one more speeds things up; with two hundred in line, one more changes nothing.
Vmax The maximum rate when the enzyme is saturated. For the lab data, about 80 nmol/min (approached, never reached). ▶ 15:00
Km (Michaelis constant) The substrate concentration at which the rate is half of Vmax. It has units of concentration. ▶ 15:30
e.g. Vmax ≈ 80, so half is 40. At 1 mM the rate was 41, so Km ≈ 1 mM.
Km = [S] at half Vmax, LOW Km = binds tight ▶ 16:00✎ Your jot
Low Km = high affinity (tight binding). High Km = low affinity (loose binding). Low number, tight binding. ▶ 17:00
Watch out A low Km does not mean a faster enzyme. Km = affinity; Vmax = top speed. An enzyme can bind tightly (low Km) but be slow (low Vmax), or the reverse. ▶ 17:30

Beta-galactosidase rate vs substrate concentration (class lab averages)

The rate climbs steeply then levels off toward Vmax (about 80), and galactose slows it at low substrate while the curves come together at high substrate.

The numbers

Substrate concentration (mM)	No inhibitor	With galactose
0	0	–
0.25	15	–
0.5	27	–
1	41	25
2	52	–
4	63	50
8	70	–
16	74	71

The Michaelis–Menten equation

Plug in Vmax, Km and [S] to get the rate; it never exceeds Vmax.

V = Vmax × [S] / (Km + [S]) ▶ 18:00
If [S] = Km: V = Vmax × Km / (2 Km) = Vmax / 2, matching the definition of Km. ▶ 18:30
e.g. Lab check: [S] = 4 mM = 4 × Km, so V = 80 × 4/(1 + 4) = 64. Measured: 63. ▶ 19:00
At very high [S] (e.g. 100 × Km), Km barely matters, [S]/(Km + [S]) ≈ 1, and V ≈ Vmax: the flat part of the curve.
Exam skills: given Km and Vmax, find the rate at a given [S]; given a curve, read off Vmax and Km. ▶ 20:00
e.g. Practice: Vmax = 200 µmol/min, Km = 5 mM.
[S] = 5 mM = Km → half of Vmax = 100 µmol/min. ▶ 22:30
[S] = 15 mM = 3 × Km → 200 × 15/20 = 150 µmol/min (three quarters of Vmax). ▶ 23:00
[S] = 45 mM → 200 × 45/50 = 180 µmol/min (nine tenths of Vmax).
Diminishing returns: tripling [S] from 5 to 15 gained 50; tripling again to 45 gained only 30. That is saturation in numbers. ▶ 23:30
Memory tip Shortcut from the class examples: if [S] = n × Km, then V = n/(n + 1) × Vmax. ▶ 23:00
Watch out Common mistakes: computing Vmax/Km, or forgetting to add Km to [S] on the bottom. If your rate is bigger than Vmax, you made a mistake. ▶ 24:00

Temperature and pH

Each enzyme has an optimum; too far past it, the enzyme loses its shape.

An enzyme's shape depends on weak bonds (hydrogen, ionic, hydrophobic). Anything that disrupts them can change the active site. ▶ 24:30
Raising temperature makes molecules collide more often and harder, so the reaction speeds up. Rough rule for many reactions: rate about doubles per 10 degrees, but only up to a point. ▶ 25:00
Denaturation The weak bonds holding the enzyme's shape break, the active site loses its shape and the enzyme unfolds. It stops working and usually doesn't recover. ▶ 25:30
The curve is not symmetric: it climbs slowly (molecules moving faster) and falls off a cliff (enzyme being destroyed). ▶ 26:00
e.g. Thermus aquaticus lives in Yellowstone hot springs; its DNA polymerase (Taq polymerase) works best around 75 °C. It makes PCR possible because it survives repeated heating above 90 °C. ▶ 26:30
pH changes the charges on active-site amino acids, so the substrate doesn't bind right; extreme pH denatures the enzyme. Each enzyme has an optimum pH suited to where it works. ▶ 27:00
Put pepsin at pH 8 and it basically stops working. ▶ 28:00

Enzyme	Where it works	Optimum pH
Pepsin (digests protein)	Stomach	About 2 (very acidic)
Salivary amylase (digests starch)	Mouth	About 7 (neutral)
Trypsin (digests protein)	Small intestine	About 8 (slightly basic)

Activity of a typical human enzyme vs temperature

Activity climbs gradually to a peak at 37 °C, then crashes as the enzyme denatures.

The numbers

Temperature (°C)	Typical human enzyme
10	25
20	45
30	75
37	100
42	90
50	40
60	5

Cofactors and coenzymes

Non-protein helpers some enzymes need to work.

Cofactor A non-protein helper an enzyme needs. Some are metal ions such as zinc, iron or magnesium. ▶ 28:00
e.g. Carbonic anhydrase helps remove carbon dioxide from blood. It has a zinc ion in its active site and doesn't work without it. About a million reactions per second. ▶ 28:30
Coenzyme A cofactor that is an organic molecule; many come from vitamins.
e.g. NAD⁺ (key in cellular respiration) is made from niacin, vitamin B3. Vitamins are parts for your enzymes. ▶ 29:00

Enzyme inhibitors

Competitive inhibitors can be outcompeted; noncompetitive and irreversible ones can't.

Competitive inhibitor Looks like the substrate and binds the active site, competing for the same spot. ▶ 29:30
With enough substrate, the substrate wins most of the time: Vmax stays the same, but more substrate is needed for half speed, so apparent Km goes up. ▶ 30:00
competitive: more substrate wins ▶ 30:30✎ Your jot
e.g. Lab: galactose (a product that resembles the substrate) is a competitive inhibitor. Rate at 1 mM fell 41 → 25; at 4 mM 63 → 50; at 16 mM only 74 → 71. Big effect at low substrate, almost none at high. ▶ 31:00
e.g. Methanol poisoning: liver alcohol dehydrogenase turns methanol into formaldehyde, then formic acid, which can cause blindness or death. Ethanol competes for the same active site, slowing this so the body can clear methanol. Today fomepizole blocks the enzyme more cleanly. ▶ 31:30
Noncompetitive inhibitor Binds somewhere other than the active site and changes the enzyme's shape so it works less well. More substrate doesn't help: Vmax goes down, Km stays the same (pure noncompetitive). ▶ 32:30
Reading an inhibition graph: look at the high-substrate end. Curves come together → competitive. Inhibited curve levels off lower → noncompetitive. ▶ 33:30
Irreversible inhibitor Binds with covalent bonds and doesn't let go; the enzyme is out for good and the cell must make new ones.
e.g. Aspirin permanently attaches a chemical group to COX (cyclooxygenase), which makes molecules involved in pain and inflammation. Ibuprofen also blocks COX but reversibly, competing for the active site. Aspirin's effect on platelets lasts days because platelets can't make new enzyme. ▶ 34:00

Feature	Competitive	Noncompetitive
Where it binds	Active site	Somewhere else on the enzyme
Vmax	Stays the same	Goes down
Km	Goes up (apparent)	Stays the same
Overcome by more substrate?	Yes	No
Graph at high substrate	Curves come together	Inhibited curve levels off lower

Allosteric regulation and feedback inhibition

Cells switch enzymes on and off through a second binding site.

Cells control their enzymes so they don't all run at full speed all the time. ▶ 34:30
Allosteric site A second binding site separate from the active site (allo = other). An activator binding there switches the enzyme on; an inhibitor switches it off, by shifting it between active and inactive shapes.
Feedback inhibition The final product of a pathway binds the allosteric site of the pathway's first enzyme and shuts it off. ▶ 35:00
When product runs low, it lets go and the pathway turns back on, like a thermostat. No energy wasted making what the cell already has. ▶ 35:30
e.g. Threonine → isoleucine takes five steps. Isoleucine inhibits the first enzyme, threonine deaminase, stopping the line at step one. ▶ 36:00
Allosteric enzymes often give a sigmoidal (S-shaped) curve: flat at low substrate, then a steep rise, then level. Like a light switch instead of a dimmer. Recognize the shape; no calculations needed. ▶ 36:30

Coming up

Problem set 4 due Friday at 5 p.m., submitted on the course site (not in class). ▶ 0:00
Question 3 is the Michaelis–Menten problem, similar to today's lab data work. ▶ 38:00
Exam 2: Thursday, October 15, in the usual room during normal class time. Covers chapters 6, 7 and 8 (energy, enzymes, cellular respiration). ▶ 0:00
Review session the Tuesday before the exam; room to be posted. ▶ 0:30
For next class: read chapter 8, sections 1 and 2 (glycolysis). ▶ 38:00
Office hours: Wednesday 2–4.

Check yourself

Why can a reaction with a large negative ΔG still be extremely slow?
What does an enzyme change about a reaction, and what does it never change?
An enzyme has Vmax = 200 µmol/min and Km = 5 mM. What is the rate at 15 mM substrate?
Enzyme A has a lower Km than enzyme B. Which binds its substrate more tightly, and is A necessarily faster?
Why does enzyme activity drop so sharply above the optimum temperature?
On a rate vs substrate graph, how do you tell a competitive inhibitor from a noncompetitive one?

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