Enzyme Kinetics Virtual Lab
Watch enzymes catch and cut their substrate, then measure it: change temperature, pH and inhibitors, read the rate in a spectrophotometer and find Km and Vmax from your own data.
The iodine method needs no instrument: a drop goes on the spotting tile every 10 s. Watch the Bench view.
Your enzyme
Lab notebook
Every run you record is written here with its settings and its initial rate. Export a CSV, or turn the session into a PDF lab report.
| # | What | Settings | Result |
|---|
Concept library
Click "New problem" to begin.
Click "Start quiz" for 5 questions on Michaelis–Menten kinetics, linear plots, inhibition, temperature and pH, and measuring rates.
1 Overview
Simulate shows the lab on the left and the graphs on the right. The lab has two views: Molecules (what happens in the tube) and Bench (the apparatus a real lab would use: a spectrophotometer, or a water bath and an iodine spotting tile for amylase). Pick an enzyme in the top bar. Experiment has six practicals with a lab report; Quiz has practice questions and a 5-question quiz.
2 The Molecules view
- Each shape is many molecules: blue enzymes with an active site, teal and white substrate, glowing products, and pink or orange inhibitors. It is an illustrative model, not to scale and slowed down, but how often each enzyme binds and turns over follows the real rate law.
- The active site closes around a bound substrate (induced fit). Above the optimum temperature the enzymes loosen and then unfold; a denatured enzyme stays gray.
- The two badges on each active site are its catalytic groups: green when their charge suits catalysis, red when the pH is wrong.
- Tap an enzyme to follow it and read what it is doing. 🏷 Labels shows or hides the labels; each label stays on one molecule and fades when it no longer applies. ✨ Show me how plays a one-minute film with a moving camera, from binding to denaturation; press it again to stop.
3 Running a reaction
- Set the substrate, enzyme, temperature and pH in the panels under the lab. In the Bench view, Blank the spectrophotometer, then press ▶: the absorbance is recorded and plotted on Progress.
- When the run ends, its initial rate (the slope of the first 10–30 s, divided by ε × path length) is recorded as a trial; press ✓ Record v₀ to record it earlier. ↺ starts a fresh tube; [S] series measures seven concentrations at once.
- Heat or extreme pH keeps damaging the enzyme while it waits: a fresh tube brings new enzyme.
4 The graphs
v₀–[S] plots your trials with a Michaelis–Menten fit (Km and Vmax with standard errors). Linear draws Lineweaver–Burk, Eadie–Hofstee or Hanes–Woolf plots, with error bars that show how 1/v₀ stretches errors. Temp and pH show the enzyme's profile (Compare adds other enzymes). Each data set has its own color and marker shape. The model curves, the fit and the tangent are learning aids, switched in the View panel.
5 Inhibitors
In the Inhibitor panel choose competitive, uncompetitive, noncompetitive or mixed, then set [I] and Ki (and α = Ki′/Ki for mixed). Run a [S] series with and without the inhibitor and compare Km and Vmax on v₀–[S] and the Lineweaver–Burk plot.
6 Experiment mode and the lab report
Six labs: amylase and pH (AQA required practical), temperature and the optimum, Km and Vmax of β-galactosidase, an unknown inhibitor, a Beer–Lambert calibration, and pepsin against trypsin. Each shows its aim, what you need and a safety note; the method ticks off as you work, and a conclusion question ends it. Then create a PDF lab report with your trials, a graph and the working.
7 Quiz and units
The practice quiz covers Michaelis–Menten, linear plots, inhibition, temperature and pH, spectrophotometry and vocabulary, each with a worked solution. SI | US switches temperatures between °C and °F; concentrations, rates and wavelengths are metric in every lab, so they do not change.
8 Keyboard
- Space starts or pauses the reaction, R records v₀, N starts a fresh tube, M switches Molecules and Bench, 1–5 pick a graph, S opens Status, Esc closes it. Right-click a view to save it as an image.
Enzyme kinetics simulator: how fast an enzyme works, and why
Enzyme kinetics is the study of how fast an enzyme converts its substrate into product, and of what speeds it up or slows it down. This free enzyme kinetics simulator lets you watch it happen at the scale of molecules and then measure it as a real lab would: with a spectrophotometer, initial rates and a Michaelis–Menten plot. Every number comes from one rate law and published constants for real enzymes, so what you see in the animation and what you measure on the graph always agree.
- Enzymes
- β-galactosidase, salivary, fungal and heat-stable amylase, pepsin, trypsin, acetylcholinesterase, catalase and a custom enzyme
- Variables
- Substrate and enzyme concentration, temperature (0–100 °C), pH (1–14), four kinds of inhibitor
- Measurements
- Progress curves, initial rates, Michaelis–Menten fit, Lineweaver–Burk, Eadie–Hofstee, Hanes–Woolf
- Modes
- Simulate, Experiment (6 practicals with a PDF report) and Quiz
- Price
- Free, no account or download; works on Chromebooks, tablets and phones
How enzymes work: the active site and induced fit
An enzyme is a protein folded into a precise three-dimensional shape. A small pocket, the active site, matches its substrate in shape and in the pattern of charges on its amino acids, which is why each enzyme is specific. When the substrate binds, the enzyme closes around it (induced fit), straining the bond that will break and lowering the activation energy. The products leave and the enzyme is unchanged, ready to work again: E + S ⇌ ES → E + P.
Michaelis–Menten kinetics: Km, Vmax and kcat
At low substrate concentration most active sites are empty, so doubling [S] nearly doubles the rate. At high [S] almost every active site is busy and the rate levels off at Vmax. The curve follows the Michaelis–Menten equation:
v₀ = Vmax [S] / (Km + [S]), with Vmax = kcat × [E]
Km is the substrate concentration that gives half of Vmax: a low Km means the enzyme works well even when substrate is scarce. kcat, the turnover number, is how many substrate molecules one active site converts each second, and kcat/Km measures catalytic efficiency. The equation holds for the initial rate, measured before the substrate runs low, which is why every run in the simulator reports v₀ from the first straight part of the progress curve.
| Enzyme | Substrate | Km | kcat (s⁻¹) | kcat/Km (M⁻¹ s⁻¹) |
|---|---|---|---|---|
| Acetylcholinesterase | acetylcholine | 0.09 mM | 1.4 × 10⁴ | 1.6 × 10⁸ |
| Catalase | hydrogen peroxide | 1.1 M | 4 × 10⁷ | 4 × 10⁷ |
| β-Galactosidase (E. coli) | ONPG | 0.95 mM | ≈ 300 | ≈ 3 × 10⁵ |
The Lineweaver–Burk plot, and why curve fitting is better
Plotting 1/v₀ against 1/[S] turns the curve into a straight line whose y-intercept is 1/Vmax and whose x-intercept is −1/Km. It is easy to read and shows inhibition patterns clearly, but taking reciprocals stretches the errors of the slowest, low-[S] points, which then pull hardest on the line. The simulator draws the same ±3 % error on every point so you can see the bars balloon, and it fits the curve itself by nonlinear least squares, the method used in research. Eadie–Hofstee (v₀ against v₀/[S]) and Hanes–Woolf ([S]/v₀ against [S]) plots are there to compare.
Types of enzyme inhibition
| Type | Binds | Km | Vmax | Lineweaver–Burk lines | Example |
|---|---|---|---|---|---|
| Competitive | the active site (free enzyme) | rises | same | meet on the y-axis | malonate on succinate dehydrogenase |
| Noncompetitive | another site, E and ES equally | same | falls | meet on the x-axis | Hg²⁺ and Ag⁺ binding –SH groups |
| Uncompetitive | the ES complex only | falls | falls | parallel | lithium on inositol monophosphatase |
| Mixed | E and ES, unequally | changes | falls | meet off the axes | many allosteric inhibitors |
Competitive vs noncompetitive inhibition
A competitive inhibitor looks like the substrate and occupies the active site, so enough substrate out-competes it: Vmax is unchanged, but the apparent Km rises by the factor 1 + [I]/Ki. A noncompetitive inhibitor binds elsewhere and changes the active site's shape, so no amount of substrate helps: Vmax falls while Km stays the same. Both are reversible, despite what some revision sites say; irreversible inhibitors such as nerve agents on acetylcholinesterase are a separate class. The Experiment mode ends with an unknown inhibitor X for you to identify from your own data.
Effect of temperature on enzyme activity
Warming speeds up collisions and the catalytic step: below the optimum the rate roughly doubles for every 10 °C rise (Q10 ≈ 2). Too hot, and the bonds that hold the enzyme's folded shape break: the active site is lost and the enzyme is denatured, usually for good. Cold only slows an enzyme down. The simulator uses the Equilibrium Model of Daniel and Danson, in which the enzyme flips between an active and an inactive form before it unfolds irreversibly, so the optimum depends on how long you measure: about 47 °C for a 20-second initial rate of β-galactosidase, but nearer 42 °C over 10 minutes. Enzymes from thermophiles, such as Taq polymerase (optimum 75–80 °C, half-life about 40 minutes at 95 °C) and the heat-stable amylase used to liquefy starch, show how far the optimum can move.
Effect of pH on enzyme activity
pH changes the charges on the amino acids of the active site. Each enzyme needs one catalytic group ionized and another protonated, so it works over a narrow range and its optimum suits where it works. There is no single "enzyme pH curve":
| Enzyme | Where it works | Optimum pH |
|---|---|---|
| Pepsin | stomach | 1.5–2.5 |
| Fungal amylase (Aspergillus) | AQA practical kit | about 5 |
| Salivary amylase | mouth | 6.7–7.0 |
| Trypsin | small intestine | 7.8–8.7 |
The amylase and pH required practical (and why it peaks near pH 5)
In AQA GCSE Biology required practical 5, amylase digests starch at 35 °C in buffers of different pH; every 10 seconds a drop of the mixture goes into iodine on a spotting tile, and the time until it no longer turns blue-black is recorded (rate = 1000 ÷ time). AQA's method uses a fungal amylase, and its own sample results are 29 s at pH 5, 46 s at pH 6, 160 s at pH 7 and over 300 s at pH 8. So the fastest time is near pH 5, not 7, a result that puzzles many classes. The simulator's fungal amylase reproduces those times; switch to salivary amylase to see the optimum move to about 7.
Measuring enzyme activity with a spectrophotometer
Many enzyme assays use a substrate that releases a colored product. β-Galactosidase cuts colorless ONPG into yellow o-nitrophenol, which absorbs at 420 nm. By the Beer–Lambert law, A = ε l c, so the rise in absorbance per minute divided by ε × l (4.5 mM⁻¹ cm⁻¹ × 1 cm) is the rate. Blank the instrument on the mixture before the enzyme goes in, read at the product's λmax, keep the absorbance below about 2, and use a quartz cuvette in the ultraviolet: catalase is followed by the fall in hydrogen peroxide at 240 nm, where glass and plastic are opaque.
Experiments and course links
| Lab | What you find out | Course links |
|---|---|---|
| Amylase and pH | The optimum pH from iodine times | AQA GCSE RP5; Edexcel GCSE |
| Temperature and the optimum | Optimum, denaturation, why assay time matters | AP Biology 3.3; AQA A-level RP1 |
| Km and Vmax of β-galactosidase | Michaelis–Menten constants and kcat | AP Biology 3.2; IB C1.1; college biochemistry |
| Unknown inhibitor | Competitive, noncompetitive, uncompetitive or mixed | AP Biology 3.3; IB HL C1.1.14–15 |
| Beer–Lambert calibration | λmax, ε and an unknown concentration | A-level practical skills; Edexcel A CP4 |
| Pepsin vs trypsin | How pH optima match the gut | GCSE digestion; Edexcel B CP1; NGSS HS-LS1-3 |
Who uses this enzyme kinetics simulator?
High school students studying enzymes for AP Biology, NGSS, GCSE, A-level and IB; college students meeting Michaelis–Menten kinetics in introductory biochemistry; and teachers who want a free, no-login virtual enzyme lab for a classroom screen, Chromebooks or homework, with a lab report at the end. It is an educational model: real enzymes vary with their source, substrate and assay conditions.
Frequently asked questions
How do you find Km and Vmax from a Lineweaver–Burk plot?
Plot 1/v₀ (y-axis) against 1/[S] (x-axis) and draw the best straight line. The y-intercept is 1/Vmax, so Vmax = 1 ÷ y-intercept. The x-intercept is −1/Km, so Km = −1 ÷ x-intercept (or Km = slope × Vmax, because the slope is Km/Vmax). For example, a y-intercept of 0.02 min/µM gives Vmax = 50 µM/min, and an x-intercept of −0.5 mM⁻¹ gives Km = 2 mM. Because taking reciprocals magnifies the errors of the slowest points, researchers now fit the Michaelis–Menten curve directly by nonlinear regression and use the double-reciprocal plot mainly to show the pattern of inhibition.
What is the difference between competitive and noncompetitive inhibition?
A competitive inhibitor resembles the substrate and binds the active site, so it competes with the substrate: adding more substrate overcomes it. Vmax is unchanged but the apparent Km rises (by the factor 1 + [I]/Ki), and Lineweaver–Burk lines meet on the y-axis. A noncompetitive inhibitor binds a different site on the enzyme, to the free enzyme and to the enzyme–substrate complex equally, and changes the active site's shape. Extra substrate cannot displace it: Vmax falls, Km stays the same, and the lines meet on the x-axis. Both kinds are reversible; heavy-metal ions are a classic noncompetitive example, though some bind irreversibly.
Why does enzyme activity decrease at high temperature?
Up to the optimum, heat makes molecules move faster, so enzyme and substrate collide more often and with more energy, and the rate roughly doubles for every 10 °C rise. Above the optimum, the extra vibration breaks the hydrogen bonds and ionic bonds that hold the enzyme's folded shape. The active site loses its shape, the substrate no longer fits, and the enzyme is denatured, usually permanently. The optimum is not a fixed number: the longer the enzyme is kept warm, the more of it is lost, so a 10-minute assay gives a lower optimum than a 20-second initial rate. Low temperatures only slow an enzyme down; they do not denature it.
Explore related simulators
Follow amylase, pepsin and trypsin through the gut, organ by organ, in the Human Digestive System Simulator. See enzymes at work inside a cell, in its mitochondria, ribosomes and chloroplasts, with the Virtual Living Cell, and find out how the enzymes of the Calvin cycle set the rate of photosynthesis in the Photosynthesis Lab. The Interactive Codon Chart and DNA to Protein Translator shows how a gene spells out the amino acids an enzyme is folded from.
Sources: Berg et al., Biochemistry 9th edition, chapter 8; Nelson & Cox, Lehninger Principles of Biochemistry 8th edition, chapter 6 and Table 6-8; OpenStax Biology 2e, section 6.5; Daniel & Danson, Trends in Biochemical Sciences 35 (2010); AQA GCSE Biology (8461) practicals handbook, required practical 5; Beers & Sizer, J. Biol. Chem. 195 (1952); Riddles et al., Methods Enzymol. 91 (1983); Sigma-Aldrich ONPG product information. An educational model: not for laboratory or clinical use.
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