BIOCHEMICAL CALCULATIONS (STH 427)

ENZYME KINETICS
Enzymes are biological catalysts which act to increase the rate of a reaction without being
used up or changed themselves. They are specific to one type of reaction and one, or a small
number of, closely related reactants known as substrates. Enzymes are a vital component of
the cell as without them, many biological reactions would be too slow to sustain life. Enzyme
kinetics is the study of enzyme reactions rates and the conditions which affect them.

Enzyme Structure

Enzymes are proteins and usually have a globular tertiary structure. Their structure is highly
specific to the reaction they catalyse, and hence the reactants involved, due to the presence of
an active site where the reaction itself occurs. This is a small cleft within the enzyme with a
specific amino acid structure allowing the substrate to bind and form the enzyme-substrate
complex (ES), which is held together by weak bonds to allow dissociation of the complex
when the reaction is finished. The rest of the enzyme acts as a scaffold, bringing these key
amino acids together.

The active site is complementary to its specific substrate’s shape. There are two main models
for how this interaction occurs:

 Lock and key model – the active site is a perfect fit for the substrate and does not
require any changes for it to bind.
 Induced fit model – the active site is almost complementary to the substrate but when
it binds, the enzyme undergoes conformational changes to make its active site’s shape
better fit. This theory is more generally accepted than the lock and key model.

Enzymes have an optimum temperature and pH at which they work best, which varies
depending on the enzyme’s function and both cellular and organ location. Changes in pH can
alter critical ionisation states, while changes in temperature can disrupt important bonds,
affecting the enzyme’s structure and therefore function. If exposed to severe changes in
temperature and/or pH, the shape of the active site may change. This is referred to as enzyme
denaturation and means the enzyme will no longer be able to bind its substrate or carry out its
biological function.

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Enzyme Function

Enzymes provide an alternative pathway for a reaction, which has a lower activation

energy (Ea) – the minimum energy input needed for a reaction to occur and convert the
substrates into products.

The transition state is a molecular intermediate between the substrate and its product, through
which the reaction passes. For example, in the equation below, X is the transition state. This
transition state has a higher free energy than both the substrate and its product, however, the
transition state is stabilised upon the addition of an enzyme.

Substrate → X → Product

Upon weak substrate binding, the enzyme’s active site changes conformation such that it fits
the transition state better than the initial substrate, hence has a higher affinity for this
transition state. This reduces the activation energy required to reach it. Therefore, when the
substrate binds to the active site, it is encouraged to continue the reaction and is converted
into the transition state, and ultimately the final product of the reaction. This energetically
favourable process allows more substrate molecules to be converted into products in a given
period of time.

Since the transition state has a high energy, it is unstable and can only exist transiently. It
spontaneously converts into the more stable product, which has a lower energy. The
enzyme’s active site has a low affinity for this product, so it dissociates and is released.

Rate-limiting Steps
The rate-limiting step of any reaction is its slowest step, and this is what sets the pace of the
entire reaction. In enzymatic reactions, the conversion of the enzyme-substrate complex to
the product is normally rate-limiting. The rate of this step (and therefore the entire enzymatic
reaction) is directly proportional to the concentration of the enzyme-substrate complex.

The concentration of the ES complex changes as the reaction progresses, and therefore the
rate of product formation also changes accordingly. When the reaction
reaches equilibrium (steady state phase), the ES concentration (and therefore the rate of
reaction) remains relatively constant.

Reaction Kinetics
When an enzyme is added to a substrate, the reaction that follows occurs in three stages with
distinct kinetics:

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The pre-steady state phase is very short, as equilibrium is reached within microseconds.
Therefore, if you measure the rate in the first few seconds of a reaction, you will be
measuring the reaction rate in the steady state. This is the rate used in Michaelis-Menten
Kinetics.

Michaelis-Menten Kinetics

Michaelis-Menten kinetics is a model of enzyme kinetics which explains how the rate of an
enzyme-catalysed reaction depends on the concentration of the enzyme and its substrate.
Let’s consider a reaction in which a substrate (S) binds reversibly to an enzyme (E) to form
an enzyme-substrate complex (ES), which then reacts irreversibly to form a product (P) and
release the enzyme again.

S + E ⇌ ES → P + E

Two important terms within Michaelis-Menten kinetics are:

 Vmax – the maximum rate of the reaction, when all the enzyme’s active sites are
saturated with substrate.
 Km (also known as the Michaelis constant) – the substrate concentration at which the
reaction rate is 50% of the Vmax. Km is a measure of the affinity an enzyme has for its
substrate, as the lower the value of Km, the more efficient the enzyme is at carrying out
its function at a lower substrate concentration.

The Michaelis-Menten equation for the reaction above is:

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This equation describes how the initial rate of reaction (V) is affected by the initial substrate
concentration ([S]). It assumes that the reaction is in the steady state, where the ES
concentration remains constant.

When a graph of substrate concentration against the rate of the reaction is plotted, we can see
how the rate of reaction initially increases rapidly in a linear fashion as substrate
concentration increases (1st order kinetics). The rate then plateaus and increasing the
substrate concentration has no effect on the reaction velocity, as all enzyme active sites are
already saturated with the substrate (0 order kinetics).

Graph of rate of reaction against substrate concentration, demonstrating Michaelis-Menten

kinetics, with Vmax and Km highlighted.

This plot of rate of reaction against substrate concentration has the shape of a rectangular
hyperbola. However, a more useful representation of Michaelis–Menten kinetics is a graph
called a Lineweaver–Burk plot, which plots the inverse of the reaction rate (1/r) against the
inverse of the substrate concentration (1/[S]). The equation used to generate this plot is given
by:

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This produces a straight line, allowing for the easier interpretation of various quantities and
values from the graph. For example, the y-intercept of the graph is equivalent to the Vmax.
The Lineweaver-Burk plot is also useful when determining the type of enzyme
inhibition present by, comparing its effect on Km and Vmax.

Different types of enzyme inhibition as shown on a Lineweaver-Burk plot

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Enzyme Kinetics (Questions and solutions)

1 What is plotted on the x and y axes on a Lineweaver-Burk plot? Show how to derive
the equation for the plot from the equation

and explain how Vmax and KM can be found from the graph's intercepts. Hint: A Lineweaver-
Burk plot is also sometimes called a double reciprocal plot.

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2 Derive the Michaelis-Menten equation by assuming rapid equilibrium.

Solution

3 Prove that Ks equals the concentration S when the initial rate is half its maximum
value.

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4 A solution initially contains a catalytic amount of an enzyme with K M = 1.5 mM, 0.25
M of substrate, and no product. After 45 seconds, the solution contains 25 µM of product.
Find Vmax and the concentration of product after 2.0 minutes.

Hint: [S] >> KM

Solution

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5 A particular enzyme at a research facility is being studied by a group of graduate
students. This enzyme has a Km value of 5.0 X 10-6 M. The students study this enzyme with
an initial substrate concentration of 0.055 M. At one minute, 7 µM of product was made.
What is the amount of product produced after 5 minutes. What is the Vmax ?

is formed

Solution

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8 Neutral sphingomyelinase 2 converts sphingomyelin into ceramide and

phosphocholine. Assume its Vmax is 35 µM min-1. When you provide 3 x 10-5 M of
sphingomyelin, you observe an initial velocity of 6.0 µM min-1. Calculate the KM for this
reaction

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