Table of Contents
Enzyme activity describes how effectively and how fast an enzyme catalyzes its specific reaction under given conditions. It is a measurable quantity: we can ask how many substrate molecules are converted into product per unit time and under which conditions this happens best.
In this chapter, the focus is on:
- how enzyme activity is defined and measured,
- how activity changes with substrate concentration,
- what is meant by $V_\text{max}$, $K_m$ and turnover number,
- how inhibitors affect enzyme activity.
(Details on how temperature, pH, concentration, and regulation influence activity are covered in the following subchapters.)
What Is Enzyme Activity?
Enzyme activity is the rate at which an enzyme converts substrate(s) into product(s). It is usually expressed as:
- Amount of product formed per unit time, or
- Amount of substrate consumed per unit time.
Common biological units:
- Enzyme unit (U):
1 U is the amount of enzyme that converts 1 micromole (\ \mu\text{mol}$) of substrate per minute under defined conditions (temperature, pH, buffer, etc.). - Catal (SI unit):
$\ \text{kat} = 1\ \text{mol·s}^{-1}$$
(1 mol of substrate converted per second).
In practice, biologists more often use U or activities “per volume” or “per mg protein” (e.g. U/mL, U/mg protein).
Two levels of description:
- Total (absolute) activity
How much catalysis a given sample can carry out in total, e.g. “500 U in this extract”. - Specific activity
Activity per amount of total protein, e.g. U per mg protein.
This is important to compare how “pure” or “efficient” an enzyme preparation is: the higher the specific activity, the more of the protein present is the active enzyme.
Measuring Enzyme Activity
To measure enzyme activity, you need:
- A known amount of enzyme.
- A known amount of substrate.
- Defined conditions (temperature, pH, ionic strength, cofactors).
Typical experimental steps:
- Mix enzyme and substrate in a buffer.
- Let the reaction run for a defined short time.
- Stop the reaction (e.g. by changing pH, heating, or adding an inhibitor).
- Measure the amount of product formed or substrate consumed.
Common measurement methods:
- Spectrophotometric assays:
Many reactions change the absorbance of light at a specific wavelength. Example: conversion of NADH (which absorbs at 340 nm) to NAD⁺ (which does not) can be followed by a decrease in absorbance:
$$A_{340} \downarrow \quad \Rightarrow \quad \text{reaction progress}.$$ - Colorimetric assays:
A colored product forms during or after the reaction, and its intensity reflects product concentration. - Fluorometric assays:
A fluorescent substrate or product changes fluorescence as the reaction proceeds. - Radioactive or labeled substrates:
Radioactively or fluorescently labeled substrates allow sensitive measurement, especially at low concentrations.
From the change in concentration over time, we calculate the initial reaction rate $v_0$ (velocity at the start of the reaction), when the substrate concentration has not yet changed much and the enzyme is not yet affected by product build-up.
Substrate Concentration and Reaction Rate
At very low substrate concentrations, adding more substrate increases the reaction rate almost proportionally: many enzyme active sites are empty and can easily bind more substrate.
As substrate concentration rises:
- More enzymes are bound to substrate at any moment.
- The rate increases, but not indefinitely.
- At high substrate concentration, nearly all enzyme molecules are working at full speed.
Eventually, further increases in substrate concentration no longer increase the reaction rate: the enzyme is saturated.
This behavior can be summarized:
- Low [S]: $v$ rises ~linearly with substrate concentration.
- Medium [S]: $v$ increases but begins to level off.
- High [S]: $v$ approaches a maximum velocity $V_\text{max}$.
This is often described by the Michaelis–Menten equation (for many enzymes under simple conditions):
$$
v = \frac{V_\text{max} \cdot [S]}{K_m + [S]}
$$
where:
- $v$ = reaction velocity,
- $V_\text{max}$ = maximum reaction velocity at enzyme saturation,
- $[S]$ = substrate concentration,
- $K_m$ = Michaelis constant.
The Michaelis Constant $K_m$
$K_m$ is a central parameter describing enzyme activity. It is defined as the substrate concentration at which the reaction velocity is half-maximal:
$$
v = \frac{1}{2} V_\text{max} \quad \text{when} \quad [S] = K_m
$$
Biological interpretation:
- Low $K_m$: The enzyme reaches half-maximal speed at low substrate concentration.
→ The enzyme has a high affinity for the substrate. - High $K_m$: The enzyme needs higher substrate concentration to reach half-maximal speed.
→ The enzyme has a lower affinity for the substrate.
In cells, an enzyme often works at substrate concentrations around its $K_m$. This allows its activity to respond sensitively to changes in substrate concentration.
Maximum Velocity $V_\text{max}$
$V_\text{max}$ is the reaction rate when:
- All enzyme active sites are occupied by substrate.
- The enzyme is operating at maximum capacity under the given conditions.
$V_\text{max}$ depends on:
- The amount (concentration) of enzyme present.
- The intrinsic speed of the enzyme’s catalytic step.
- Experimental conditions (temperature, pH, etc.).
If you double the enzyme concentration (keeping substrate constant and high), you approximately double $V_\text{max}$.
Turnover Number ($k_\text{cat}$)
Another useful measure of enzyme activity is the turnover number $k_\text{cat}$:
- $k_\text{cat}$ = the number of substrate molecules converted into product by one enzyme molecule per second when the enzyme is fully saturated with substrate.
In equation form:
$$
k_\text{cat} = \frac{V_\text{max}}{[E]_\text{total}}
$$
where $[E]_\text{total}$ is the total enzyme concentration.
Typical values:
- Some enzymes have $k_\text{cat}$ of a few reactions per second.
- Very efficient enzymes (e.g. catalase, carbonic anhydrase) can reach up to $10^6$–$10^7$ substrate molecules per second per enzyme molecule.
A related concept is catalytic efficiency, which combines $k_\text{cat}$ and $K_m$:
$$
\text{catalytic efficiency} = \frac{k_\text{cat}}{K_m}
$$
This ratio is particularly informative at low substrate concentrations, such as those often found inside cells.
Enzyme Inhibition and Apparent Activity
Enzyme activity in real cells is rarely measured under “ideal” conditions. It is influenced by molecules that inhibit (reduce) or activate (increase) the activity of enzymes.
Here, the focus is on the basic types of inhibition and how they show up as changes in measurable activity. (Detailed cellular regulation is discussed in the dedicated regulation subchapter.)
Reversible Inhibition
Reversible inhibitors bind non-covalently and can dissociate again. Their effects on activity depend on how and where they bind.
Competitive Inhibition
- The inhibitor resembles the substrate and competes for the active site.
- Inhibitor and substrate cannot bind at the same time.
- Effect on activity:
- At low substrate concentration, inhibitor strongly reduces activity.
- If you increase substrate concentration sufficiently, you can overcome the inhibition (because substrate outcompetes the inhibitor).
In Michaelis–Menten terms:
- $V_\text{max}$ remains unchanged (can still be reached at high substrate).
- Apparent $K_m$ increases (it takes more substrate to reach half $V_\text{max}$).
Biological meaning: competitive inhibitors effectively lower the apparent affinity of the enzyme for its substrate.
Non-Competitive Inhibition (a form of allosteric inhibition)
- The inhibitor binds at a site different from the active site (an allosteric site).
- The inhibitor can bind whether or not the substrate is bound.
- Binding causes a change in enzyme conformation that reduces catalytic efficiency.
Effect on activity:
- Increasing substrate concentration does not fully overcome inhibition.
- Often:
- $V_\text{max}$ decreases (the maximal rate is lowered).
- $K_m$ remains unchanged (the substrate affinity is not necessarily affected).
Biological meaning: the enzyme’s “maximal capacity” is reduced, as if some fraction of enzymes is “turned off”.
Uncompetitive Inhibition
- The inhibitor binds only to the enzyme–substrate complex (not to the free enzyme).
- This usually occurs at a site distinct from the active site but only after substrate is bound.
Effect on activity:
- Both $V_\text{max}$ and $K_m$ decrease.
- Kinetic curves shift in a characteristic way, but this form of inhibition is less common in basic textbook examples compared to competitive and non-competitive inhibition.
Irreversible Inhibition
Irreversible inhibitors:
- Form covalent bonds with the enzyme or
- Destroy key functional groups in the active site.
As a result, the enzyme is permanently inactivated. This essentially reduces the total amount of active enzyme present.
Consequences for measured activity:
- $V_\text{max}$ decreases (less active enzyme molecules are available).
- The effect cannot be removed by washing out the inhibitor; new enzyme needs to be synthesized.
Examples (without going into mechanism details): many poisons and some drugs act as irreversible enzyme inhibitors.
Initial Rate, Reaction Progress, and Apparent Activity
When following enzyme activity over time, the reaction curve typically shows:
- Initial linear phase
Rate $v_0$ is constant; substrate is abundant, product is low, and conditions are stable. - Slowing phase
Substrate is depleted and product accumulates. The reaction rate gradually declines. - Plateau
An equilibrium (or steady state) is approached; net change in product vs. substrate is minimal.
To characterize enzyme activity, biochemists focus on the initial rate $v_0$ because it reflects the intrinsic catalytic properties of the enzyme under defined conditions, without complications from changing substrate/product levels.
Apparent activity can decrease over time for several reasons:
- Substrate depletion.
- Product inhibition.
- Denaturation or inactivation of the enzyme.
- Changes in pH or other conditions in the reaction mixture.
Distinguishing these experimental effects from true differences in enzyme properties is important when interpreting activity measurements.
Biological Relevance of Enzyme Activity
In living organisms, enzyme activity determines:
- How fast key metabolic pathways proceed (e.g. glycolysis, cellular respiration, photosynthesis).
- How quickly cells respond to changes in nutrient availability.
- How tightly flux through pathways can be controlled by regulators, inhibitors, and activators.
Although the detailed mechanisms of regulation and their physiological roles are discussed in later sections, the central idea here is:
- Enzyme activity connects enzyme structure and environmental conditions to actual metabolic rates in cells.
- Measuring and understanding activity is therefore essential to understanding metabolism as a whole.