IMAT Biology · Biomolecules and Enzymes

Temperature, pH, Substrate and Inhibitors

Enzyme rate rises with temperature until the protein denatures, peaks at an optimum pH, levels off as substrate saturates the active sites, and falls when an inhibitor blocks or distorts the enzyme.

All Biomolecules and Enzymes notes

Why this matters

Two ministry questions sit here: which enzymes can work in the same pH (2023) and what happens in competitive inhibition (2025). The older papers asked for the right rate curves with and without a competitive inhibitor (2014), what all inhibitors have in common (2017), how a drug that mimics nucleotides acts (2018), and how to read temperature and pH graphs (2022).

Concept 1 of 3: Effect of temperature and pH on enzyme activity

Warming speeds molecules up, so they meet more often and with more energy, and the rate rises. But the enzyme's shape is held by weak bonds, and too much heat shakes them apart: the active site loses its shape and activity collapses. pH works on the same weak bonds, by changing the charges on R groups, so each enzyme works only inside a range of pH around its optimum.

Definition

Temperature: below the optimum, the rate roughly doubles for every 10 °C rise. Above the optimum (about 37 to 40 °C for most human enzymes), the enzyme denatures: the active site changes shape and the rate falls steeply. Denaturation by heat is usually irreversible.
Cold does not denature: at low temperature an enzyme is simply slow, and works again when warmed.
Enzymes of organisms from hot springs (such as the Taq polymerase used in PCR) have optima above 70 °C.
pH: each enzyme has an optimum pH and a working range. Away from it, the charges on R groups change, ionic and hydrogen bonds break, and the active site changes shape. Pepsin works best at about pH 2, salivary amylase near pH 7, trypsin near pH 8.
Two enzymes can work in the same solution only at a pH inside both of their working ranges.
In an experiment on one factor, the others (pH, temperature, concentrations) are kept constant, usually at their optimum.

Condition	Effect on rate	Reason
Temperature rising below the optimum	Increases	More frequent and more energetic collisions
Temperature above the optimum	Falls steeply	Bonds holding the shape break; the enzyme denatures
Low temperature	Very slow, but recovers on warming	Few collisions; the enzyme is not denatured
pH away from the optimum	Falls on both sides	Charges on R groups change; the active site changes shape
More enzyme, with plenty of substrate	Increases in proportion	More active sites available

Practice this conceptself-check · 4 quick reps

Low temperature inactivates; high temperature denatures

Cooling slows an enzyme but leaves its shape intact, so activity returns on warming. Heating above the optimum breaks the bonds holding the shape, and this is usually permanent. Do not say an enzyme is denatured by cold.

Concept 2 of 3: Substrate concentration, Vmax and Km

With little substrate, most active sites are empty, so adding substrate fills more of them and the rate climbs almost in proportion. As substrate builds up, nearly every active site is busy all the time, and adding more cannot make the enzyme work faster. The rate levels off at a maximum set by how much enzyme there is.

Definition

At low substrate concentration the rate rises steeply; at high concentration it levels off at the maximum rate, $V_{\max}$ , because all active sites are occupied (the enzyme is saturated).
$V_{\max}$ depends on the amount of enzyme: double the enzyme, double $V_{\max}$ .
The Michaelis constant $K_m$ is the substrate concentration at which the rate is half of $V_{\max}$ .
A low $K_m$ means the enzyme reaches half speed at a low substrate concentration: it has a high affinity for its substrate.
IMAT asks this mostly in words or as a graph; the equation below shows where the curve comes from.

Michaelis-Menten equation

v = \frac{V_{\max}\,[S]}{K_m + [S]}

$v$ initial rate of reaction
$V_{\max}$ maximum rate, when all active sites are occupied
$[S]$ substrate concentration
$K_m$ substrate concentration giving half of $V_{\max}$

Worked example

An enzyme has

V_{\max} = 40\ \mu\text{mol/min}

and

K_m = 2.0\ \text{mmol/dm}^3

. Find the initial rate when the substrate concentration is 2.0 and when it is 6.0

\text{mmol/dm}^3

Practice this conceptself-check · 4 quick reps

Km is a substrate concentration, not a rate

K_m

is the substrate concentration at half the maximum rate, so its unit is a concentration. A smaller

K_m

means higher affinity, not a slower enzyme.

Concept 3 of 3: Competitive, non-competitive and irreversible inhibition

A competitive inhibitor looks like the substrate and sits in the active site, so the two compete for the same seat: flood the enzyme with substrate and the substrate wins. A non-competitive inhibitor binds somewhere else and bends the enzyme out of shape, so no amount of substrate can help. The cell also uses inhibition on purpose, letting the end product of a pathway switch off its first enzyme.

Definition

Competitive inhibitor: similar in shape to the substrate; binds reversibly to the active site, blocking the substrate. Adding more substrate overcomes it. $V_{\max}$ is unchanged; $K_m$ rises. On a rate against substrate graph, the curve rises more slowly but reaches the same plateau.
Example: drugs that mimic nucleotides block the viral enzyme reverse transcriptase, so no new phosphodiester bonds form and fewer virus particles are made.
Non-competitive inhibitor: binds to another site (often an allosteric site), changing the shape of the active site. More substrate does not overcome it. $V_{\max}$ is lowered; $K_m$ is unchanged. The curve levels off lower.
Irreversible inhibitors bind permanently, often covalently: cyanide on cytochrome oxidase, nerve agents on acetylcholinesterase, aspirin on cyclooxygenase.
End-product (feedback) inhibition: the final product of a pathway inhibits an early enzyme, usually allosterically, so the cell makes only what it needs.
The only thing every inhibitor does is reduce the rate. Not all change the active site's shape, none denature the enzyme, and they do not raise the activation energy of the catalysed reaction.

Type	Where it binds	Effect of adding more substrate	Effect on Vmax and Km
Competitive	Active site	Overcomes the inhibition	Vmax same; Km higher
Non-competitive	Another site (allosteric)	Does not help	Vmax lower; Km same
Irreversible	Usually the active site, permanently	Does not help	Enzyme molecules lost for good
End-product (feedback)	Allosteric site of an early enzyme	Depends on the product level	Pathway slows when product builds up

Practice this conceptself-check · 3 quick reps

A competitive inhibitor binds the enzyme, not the substrate

The inhibitor and the substrate compete for the same active site. The inhibitor does not attach to the substrate, and it does not block the release of products. Options describing either are wrong.

The one property all inhibitors share is a lower rate

Competitive inhibitors do not change the active site's shape; no inhibitor denatures the enzyme; none raise the activation energy of the catalysed reaction. The only statement true of every inhibitor is that it reduces the rate of the reaction.

Summary — formulas & gotchas at a glance

A revision cheat-sheet for the formulas and gotchas above. Click any concept name to jump back to its full explanation.

Formulas (1)

Substrate concentration, Vmax and Km
Michaelis-Menten equation
$v = \frac{V_{\max}\,[S]}{K_m + [S]}$

Reference tables (2)

Effect of temperature and pH on enzyme activity5 rows

Condition	Effect on rate	Reason
Temperature rising below the optimum	Increases	More frequent and more energetic collisions
Temperature above the optimum	Falls steeply	Bonds holding the shape break; the enzyme denatures
Low temperature	Very slow, but recovers on warming	Few collisions; the enzyme is not denatured
pH away from the optimum	Falls on both sides	Charges on R groups change; the active site changes shape
More enzyme, with plenty of substrate	Increases in proportion	More active sites available

Competitive, non-competitive and irreversible inhibition4 rows

Type	Where it binds	Effect of adding more substrate	Effect on Vmax and Km
Competitive	Active site	Overcomes the inhibition	Vmax same; Km higher
Non-competitive	Another site (allosteric)	Does not help	Vmax lower; Km same
Irreversible	Usually the active site, permanently	Does not help	Enzyme molecules lost for good
End-product (feedback)	Allosteric site of an early enzyme	Depends on the product level	Pathway slows when product builds up

Watch out for (4)

Low temperature inactivates; high temperature denatures
→ Effect of temperature and pH on enzyme activity
Km is a substrate concentration, not a rate
→ Substrate concentration, Vmax and Km
A competitive inhibitor binds the enzyme, not the substrate
→ Competitive, non-competitive and irreversible inhibition
The one property all inhibitors share is a lower rate
→ Competitive, non-competitive and irreversible inhibition

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