Enzymes are proteins that play a major role in the biochemical reactions happening every moment

inside our bodies - everything from digesting a bowl of ramen noodles to flexing your muscles in
front of a mirror.

Enzymes act as catalysts - meaning that they speed up the rate at which these biochemical
reactions happen.

So instead of waiting months to years for a reaction to happen, it can happen in seconds - which is
essential for life to happen.

Imagine trying to digest a single bowl of ramen for a year - you’d die of hunger before you could do
it!

Every biochemical reaction has a substrate and a product - so let’s put them on this graph called a
reaction coordinate diagram.

The X axis shows how a reaction progresses, while the Y axis shows the energy level at the different
points along the reaction.

In the beginning, we’ve got the substrate - let’s call it A - with a fair amount of free energy.

At the end of it, there’s the product - or B, which ranks lower energy-wise.

The energy of the product minus the energy of the substrate is called the energy of the reaction,
also known as Gibbs free energy, or ΔG.

Because lower energy states are preferred, a reaction spontaneously occurs when the product has
a lower free energy than the substrate - so a negative ΔG.

So let’s say we’re looking at one such spontaneously occurring reaction, but between going from
the substrate to the product there’s an intermediate transition step that has a really high energy
state.

The amount of extra energy the substrate requires to get to the transition state - so the height of
the upslope - is called the activation energy - or a ΔG‡ plus plus.

As soon as it enters the transition state, the molecule is highly unstable - and wants to go to a more
stable lower-energy molecule

It either goes back to being a substrate or to being a product.

If it’s a substrate once again, it can go back up to the transition state if there’s enough activation
energy once more, but if it becomes a product then it needs even more energy to get back to the
transition state.

That’s why over time, with millions of molecules doing this, the majority of substrate turns into
product.

Now, without an enzyme, the substrate might eventually harness enough activation energy to
enter the transition state - but enzymes help speed things up quite a bit.
Enzymes are proteins that are folded in a particular way, so that they have a pocket called the
active site on their surface.

When enzymes get involved in a reaction, the substrate binds to the active site, and together they
form an enzyme-substrate complex, and that helps stabilize the transition state.

So enzymes decrease that extra energy requirement for the reaction - graphically turning our
mountain into a hill.

Consider this analogy.

Imagine a little boy who’s nervous about getting a vaccine - he’s the substrate, and he turns into a
vaccinated child - that’s the product.

The transition state is where the needle goes in, and as you can imagine - the boy might get really
anxious and upset - a highly energetic and uncomfortable state.

In this scenario, enzymes are like adults who hold the boy and calm him down, reducing the
anxiety or energy level of the transition state and making the whole thing happen faster.

Fortunately, enzymes don’t get used up in the process.

They attach to the substrate until it turns into the product and then release the product.

As soon as they’re done, they find another substrate.

What’s more is that enzymes and substrates are like biochemical soulmates - each enzyme is
specifically designed for a particular type of substrate.

For example, amylase is an enzyme in your saliva that specifically helps break down large
carbohydrates - into smaller sugar molecules that are then further broken down by other enzymes.

Now, the rate at which enzymes catalyse biochemical reactions is called enzyme kinetics, and there
are two graphical ways to look at this.

The first, is the Michaelis Menten graph which has the concentration of the substrate, or [S], on
the X axis, and the speed, or velocity of the reaction or V, which is how much product is formed
over time, on the Y axis.

If there’s a fixed amount of enzyme, the velocity of the reaction increases as more substrate is
added - that is, until all the active sites on all of the enzyme become saturated.

At this point, adding more substrate won’t do a thing, because there’s no more enzyme to bind it -
so the speed of the reaction plateaus.

The point where the curve flattens out corresponds to the maximum velocity, or Vmax, on the Y
axis.

Now we can determine Km - which is the concentration of substrate at which the speed of the
reaction is exactly half the maximum velocity.
So we look at the Y axis, find what half of Vmax is, then we go parallel to the X axis until we reach
our reaction curve.

From there, we go straight down towards the X axis - and Km will be equal to that substrate
concentration.

The reason that Km is worth figuring out is that it inversely reflects enzyme affinity - if Km is low,
only a little substrate is needed for the reaction to skyrocket up to half of its maximum rate, so
we’re looking at an enzyme with high affinity.

On the other hand, if Km is high, then it takes a lot of substrate to get the reaction to go at half the
maximum rate - so the enzyme has low affinity for its substrate.

Fundamentally, these graphs tell us about binding.

In fact, the same graphic representation could be used to show how strongly drugs binding to
receptors, or even transcription factors binding to DNA!

So while we’re here, let’s look at how Vmax or Km can increase or decrease.

Vmax depends on the number of enzymes that can catalyse the reaction - so increasing or
decreasing the number of enzymes will increase and decrease Vmax.

One way to increase Vmax is induction, which is when there’s an increase in gene expression of an
enzyme.

The opposite - a decrease in Vmax - might happen if there’s repression or silencing of gene
expression.

If we’re talking about membrane receptors or transporters, up-regulation or down-regulation of

Finally, a drug might block enzymes, transporters or receptors non-competitively and cause a
decrease in Vmax.

With non-competitive inhibition, the inhibitory molecule binds irreversibly to the enzyme active
site, or reversibly to a different place called the allosteric site.

The net effect is that substrates can no longer bind to the active site and it is as if the number of
available enzymes had decreased.

So the effects of non-competitive inhibition are similar to what happens when there’s less enzyme
around, decreasing Vmax.

Now let’s look at Km, which varies according to enzyme affinity.

Affinity depends on the shape of the enzyme, and how well the substrate fits into the active site.

So, let’s say we alter the enzyme in a way that makes it bind more substrate - one way to do this is
when a molecule called an activator binds to the enzyme and increases that enzyme’s affinity for
the substrate, lowering the Km.
Conversely, competitive inhibition can be used to decrease the affinity of the enzyme.

A competitive inhibitor binds to the active site instead of the substrate, and usually doesn’t get
metabolized - blocking the enzyme.

Now, since this is a reversible process, adding more substrate means that it’s possible to eventually
outcompete and displace the inhibitor.

But this increases the Km.

Now, practically speaking, a common way to change the shape of an enzyme is through
phosphorylation by kinases or dephosphorylation by phosphatases, and many processes are
regulated this way in our cells.

In fact, the terms desensitization and sensitization refer to changes in a receptor’s shape which
decrease or increase its affinity for a drug.

Now, let’s switch gears and look at the Lineweaver-Burke plot.

This graph is based on the Michaelis Menten equation - which states that the initial speed of the
reaction - or V0 - is equal to the concentration of substrate - [S] times the maximum velocity -
Vmax, over the concentration of substrate + Km.

So here’s where things get interesting - to get to our Lineweaver Burke plot, we’re writing this
equation down as a double reciprocal.

So flipping the whole thing upside down, we get that 1 over V0 is equal to Km over Vmax plus 1
over Vmax plus 1 over the concentration of substrate - [S].

Now we can use this equation on the plot: 1/V0 goes on the Y axis and 1/[S] goes on the X axis.

Conveniently enough, after flipping the equation upside down, Km/Vmax is represented as a
straight line, that intercepts both the Y and the X axes.

Y intercept is equal to 1/Vmax, and X intercept - since it happens negative to the origin of the axes
- is -1/Km.

Thus, the x- and y-intercepts can be used to quickly identify Km and Vmax.

The only trick is that, much like our reciprocal equation, now everything is upside down!

So processes like induction and upregulation which increase the Vmax, actually cause 1/Vmax to
decrease so the line representing Km/Vmax would slope lower on the graph than the control.

Conversely, processes like repression or downregulation or even non competitive inhibition which
decrease the Vmax, will increase 1/Vmax, making the line slope higher on the graph than the
control.

So interpreting a Lineweaver Burke plot goes like this.

Stimulation makes the straight line appear to go down, inhibition makes it appear to shoot higher -
it’s, well, the reciprocal!
Summary

Alright, as a quick recap.

Enzymes lower the activation energy required for the substrate to enter the transition state.

Substrate binds to the enzyme at the active site, forming an enzyme-substrate complex, and it is
released after it’s transformed into the product.

There are two main ways to look at enzyme kinetics: the Michaelis Menten graph, and the
Lineweaver Burke plot.

Both can be used to determine the two kinetic variables, Vmax, or the maximum speed of the
reaction, and Km, or the affinity of the enzyme for its substrate.