Enzymes and the active site

Enzymes as biological catalysts, activation energy, the active site, and environmental effects
on enzyme activity.

Introduction
As a kid, I wore glasses and desperately wanted a pair of contact
lenses. When I was finally allowed to get contacts, part of the deal
was that I had to take very, very good care of them, which meant
washing them with cleaner every day, storing them in a sterile
solution, and, once a week, adding a few drops of something called
“enzymatic cleaner.” I didn’t know exactly what “enzymatic cleaner”
meant, but I did learn that if you forgot you’d added it and
accidentally put your contacts in your eyes without washing them,
you were going to have burning eyes for a good fifteen minutes.

As I would later learn, all that “enzymatic” meant was that the
cleaner contained one or more enzymes, proteins that catalyzed
particular chemical reactions – in this case, reactions that broke
down the film of eye goo that accumulated on my contacts after a
week of use. (Presumably, the reason it stung when I got it in my
eyes was that the enzymes would also happily break down eye goo in
an intact eye.) In this article, we’ll look in greater depth at what an
enzyme is and how it catalyzes a particular chemical reaction.

Enzymes and activation energy

A substance that speeds up a chemical reaction—without being a
reactant—is called a catalyst. The catalysts for biochemical
reactions that happen in living organisms are called enzymes.
Enzymes are usually proteins, though some ribonucleic acid (RNA)
molecules act as enzymes too.

Enzymes perform the critical task of lowering a reaction's activation

energy—that is, the amount of energy that must be put in for the
reaction to begin. Enzymes work by binding to reactant molecules
and holding them in such a way that the chemical bond-breaking and
bond-forming processes take place more readily.

Reaction coordinate diagram showing the course of a reaction with

and without a catalyst. With the catalyst, the activation energy is
lower than without. However, the catalyst does not change the ∆G for
the reaction.
_Image modified from "Potential, kinetic, free, and activation energy: Figure 5," by OpenStax
College, Biology, CC BY 3.0._

To clarify one important point, enzymes don’t change a

reaction’s ∆G value. That is, they don’t change whether a reaction is
energy-releasing or energy-absorbing overall. That's because
enzymes don’t affect the free energy of the reactants or products.
Instead, enzymes lower the energy of the transition state, an
unstable state that products must pass through in order to become
reactants. The transition state is at the top of the energy "hill" in the
diagram above.

Active sites and substrate specificity

To catalyze a reaction, an enzyme will grab on (bind) to one or more
reactant molecules. These molecules are the enzyme's substrates.

In some reactions, one substrate is broken down into multiple

products. In others, two substrates come together to create one
larger molecule or to swap pieces. In fact, whatever type of biological
reaction you can think of, there is probably an enzyme to speed it up!

The part of the enzyme where the substrate binds is called the active
site (since that’s where the catalytic “action” happens).
 A substrate enters the active site of the enzyme. This forms the
enzyme-substrate complex.The reaction then occurs, converting the
substrate into products and forming an enzyme products complex.
The products then leave the active site of the enzyme.
Image modified from "Enzymes: Figure 2," by OpenStax College, Biology, CC BY 3.0.

Proteins are made of units called amino acids, and in enzymes that
are proteins, the active site gets its properties from the amino acids
it's built out of. These amino acids may have side chains that are
large or small, acidic or basic, hydrophilic or hydrophobic.

The set of amino acids found in the active site, along with their
positions in 3D space, give the active site a very specific size, shape,
and chemical behavior. Thanks to these amino acids, an enzyme's
active site is uniquely suited to bind to a particular target—the
enzyme's substrate or substrates—and help them undergo a
chemical reaction.
[How specific is the matching between enzyme and substrate?]

Environmental effects on enzyme function

Because active sites are finely tuned to help a chemical reaction
happen, they can be very sensitive to changes in the enzyme’s
environment. Factors that may affect the active site and enzyme
function include:

• Temperature. A higher temperature generally makes for higher

rates of reaction, enzyme-catalyzed or otherwise. However, either
increasing or decreasing the temperature outside of a tolerable
range can affect chemical bonds in the active site, making them less
 well-suited to bind substrates. Very high temperatures (for animal
enzymes, above 404040 ^{\circ}\text C∘Cdegrees, start text, C, end
text or 104104104 ^{\circ}\text F∘Fdegrees, start text, F, end text)
may cause an enzyme to denature, losing its shape and
activity.^22squared

• pH. pH can also affect enzyme function. Active site amino acid
residues often have acidic or basic properties that are important for
catalysis. Changes in pH can affect these residues and make it hard
for substrates to bind. Enzymes work best within a certain pH range,
and, as with temperature, extreme pH values (acidic or basic) can
make enzymes denature.

Induced fit
The matching between an enzyme's active site and the substrate isn’t
just like two puzzle pieces fitting together (though scientists once
thought it was, in an old model called the “lock-and-key” model).

Instead, an enzyme changes shape slightly when it binds its

substrate, resulting in an even tighter fit. This adjustment of the
enzyme to snugly fit the substrate is called induced fit.
 Illustration of the induced fit model of enzyme catalysis. As a
substrate binds to the active site, the active site changes shape a
little, grasping the substrate more tightly and preparing to catalyze
the reaction. After the reaction takes place, the products are released
from the active site and diffuse away.
Image modified from "Enzymes: Figure 2," by OpenStax College, Biology, CC BY 3.0.

When an enzyme binds to its substrate, we know it lowers the

activation energy of the reaction, allowing it to happen more quickly.
But, you may wonder, what does the enzyme actually do to the
substrate to make the activation energy lower?

The answer depends on the enzyme. Some enzymes speed up

chemical reactions by bringing two substrates together in the right
orientation. Others create an environment inside the active site
that's favorable to the reaction (for instance, one that's slightly acidic
or non-polar). The enzyme-substrate complex can also lower
activation energy by bending substrate molecules in a way that
facilitates bond-breaking, helping to reach the transition state.

Finally, some enzymes lower activation energies by taking part in the

chemical reaction themselves. That is, active site residues may form
temporary covalent bonds with substrate molecules as part of the
reaction process.

An important word here is "temporary." In all cases, the enzyme will

return to its original state at the end of the reaction—it won't stay
bound to the reacting molecules. In fact, a hallmark property of
enzymes is that they aren't altered by the reactions they catalyze.
When an enzyme is done catalyzing a reaction, it just releases the
product (or products) and is ready for the next cycle of catalysis.

Enzyme regulation
AP.BIO:
ENE-1 (EU)
,
ENE-1.G (LO)

,
ENE-1.G.2 (EK)
,
ENE-1.G.4 (EK)
Cofactors and coenzymes. Reversible, irreversible, competitive, and noncompetitive inhibitors.
Allosteric enzymes. Feedback inhibition.

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Introduction
The cells of your body are capable of making many different
enzymes, and at first you might think: great, let’s crank all of those
enzymes up and metabolize as fast as possible! As it turns out, though,
 you really don’t want to produce and activate all of those enzymes at
the same time, or in the same cell.

Needs and conditions vary from cell to cell and change in individual
cells over time. For instance, stomach cells need different enzymes
than fat storage cells, skin cells, blood cells, or nerve cells. Also, a
digestive cell works much harder to process and break down
nutrients during the time that follows a meal as compared with
many hours after a meal. As these cellular demands and conditions
changes, so do the amounts and functionality of different enzymes.

Because enzymes guide and regulate the metabolism of a cell, they

tend to be carefully controlled. In this article, we’ll take a look at
factors that can affect or control enzyme activity. These include pH
and temperature (discussed in the active site article), as well as:

• Regulatory molecules. Enzyme activity may be turned "up" or

"down" by activator and inhibitor molecules that bind specifically to
the enzyme.
• Cofactors. Many enzymes are only active when bound to non-
protein helper molecules known as cofactors.
• Compartmentalization. Storing enzymes in specific compartments
can keep them from doing damage or provide the right conditions
for activity.
• Feedback inhibition. Key metabolic enzymes are often inhibited by
the end product of the pathway they control (feedback inhibition).
In the rest of this article, we'll examine these factors one at a time,
seeing how each can affect enzyme activity.

Regulatory molecules
 Enzymes can be regulated by other molecules that either increase or
reduce their activity. Molecules that increase the activity of an
enzyme are called activators, while molecules that decrease the
activity of an enzyme are called inhibitors.

There are many kinds of molecules that block or promote enzyme

function, and that affect enzyme function by different routes.

Competitive vs. noncompetitive

In many well-studied cases, an activator or inhibitor's binding is
reversible, meaning that the molecule doesn't permanently attach to
the enzyme. Some important types of drugs act as reversible
inhibitors. For example, the drug tipranivir, which is used to treat
HIV, is a reversible inhibitor.^11start superscript, 1, end
superscript It blocks activity of a viral enzyme that helps the virus
make more copies of itself.

Reversible inhibitors are divided into groups based on their binding

behavior. We won't discuss all of the types here, but we will look at
two important groups: competitive and noncompetitive inhibitors.

• An inhibitor may bind to an enzyme and block binding of the

substrate, for example, by attaching to the active site. This is
called competitive inhibition, because the inhibitor “competes”
with the substrate for the enzyme. That is, only the inhibitor or the
substrate can be bound at a given moment.
• In noncompetitive inhibition, the inhibitor doesn't block the
substrate from binding to the active site. Instead, it attaches at
another site and blocks the enzyme from doing its job. This
 inhibition is said to be "noncompetitive" because the inhibitor and
substrate can both be bound at the same time.

Diagram illustrating competitive and noncompetitive inhibition. The

competitive inhibitor binds to the active site and prevents the
substrate from binding there. The noncompetitive inhibitor binds to
a different site on the enzyme; it doesn't block substrate binding, but
it causes other changes in the enzyme so that it can no longer
catalyze the reaction efficiently.

Competitive and non-competitive inhibitors can be told apart by how

they affect an enzyme's activity at different substrate concentrations.

• If an inhibitor is competitive, it will decrease reaction rate when

there's not much substrate, but can be "out-competed" by lots of
substrate. That is, the enzyme can still reach its maximum reaction
rate given enough substrate. In that case, almost all the active sites of
almost all the enzyme molecules will be occupied by the substrate
rather than the inhibitor.
[See a sports analogy]
• If an inhibitor is noncompetitive, the enzyme-catalyzed reaction will
never reach its normal maximum rate even with a lot of substrate.
This is because the enzyme molecules with the noncompetitive
inhibitor bound are "poisoned" and can't do their job, regardless of
how much substrate is available.
On a graph of reaction velocity (y-axis) at different substrate
concentrations (x-axis), you can tell these two types of inhibitors
apart by the shape of the curves:

This plot shows rate of reaction versus substrate concentration for

an enzyme in the absence of inhibitor, and for enzyme in the
presence of competitive and noncompetitive inhibitors. Both
competitive and noncompetitive inhibitors slow the rate of reaction,
but competitive inhibitors can be overcome by high concentrations
of substrate, whereas noncompetitive inhibitors cannot.
_Image credit: "Enzymes: Figure 3," by OpenStax College, Biology, CC BY 3.0._
Not familiar with this type of graph? No worries! The basics of
enzyme kinetics graphs article has a step-by-step walkthrough.

Allosteric regulation
Allosteric regulation, broadly speaking, is just any form of
regulation where the regulatory molecule (an activator or inhibitor)
binds to an enzyme someplace other than the active site. The place
where the regulator binds is called the allosteric site.

The left part of this diagram shows allosteric inhibition. The

allosteric inhibitor binds to an enzyme at a site other than the active
site. The shape of the active site is altered so that the enzyme can no
longer bind to its substrate.
 The right part of this diagram shows allosteric activation. The
allosteric activator binds to an enzyme at a site other than the active
site. The shape of the active site is changed, allowing substrate to
bind at a higher affinity.
_Image modified from "Enzymes: Figure 4," by OpenStax College, Biology, CC BY 3.0._

Pretty much all cases of noncompetitive inhibition (along with some

unique cases of competitive inhibition) are forms of allosteric
regulation.

However, some enzymes that are allosterically regulated have a set

of unique properties that set them apart. These enzymes, which
include some of our key metabolic regulators, are often given the
name of allosteric enzymes^22squared. Allosteric enzymes typically
have multiple active sites located on different protein subunits.
When an allosteric inhibitor binds to an enzyme, all active sites on
the protein subunits are changed slightly so that they work less well.

There are also allosteric activators. Some allosteric activators bind to

locations on an enzyme other than the active site, causing an
increase in the function of the active site. Also, in a process
called cooperativity, the substrate itself can serve as an allosteric
activator: when it binds to one active site, the activity of the other
active sites goes up.^{3}3cubed This is considered allosteric
regulation because the substrate affects active sites far from its
binding site.
[Is hemoglobin an allosteric enzyme?]

Cofactors and coenzymes

Many enzymes don’t work optimally, or even at all, unless bound to
other non-protein helper molecules called cofactors. These may be
attached temporarily to the enzyme through ionic or hydrogen
bonds, or permanently through stronger covalent bonds.Common
cofactors include inorganic ions such as iron \text
{(Fe}^{2+})(Fe2+)start text, left parenthesis, F, e, end text, start
superscript, 2, plus, end superscript, right parenthesis and
magnesium (\text {Mg}^{2+})(Mg2+)left parenthesis, start text, M, g,
end text, start superscript, 2, plus, end superscript, right parenthesis.
For example, the enzyme that builds DNA molecules, DNA
polymerase, requires magnesium ions to function.^44start
superscript, 4, end superscript

Coenzymes are a subset of cofactors that are organic (carbon-based)

molecules. The most common sources of coenzymes are dietary
vitamins. Some vitamins are precursors to coenzymes and others act
directly as coenzymes. For example, vitamin C is a coenzyme for
several enzymes that take part in building the protein collagen, a key
part of connective tissue.
Chemical structure of vitamin C, which acts as a coenzyme for several
enzymes.
Image modified from OpenStax Biology.

Enzyme compartmentalization
Enzymes are often compartmentalized (stored in a specific part of
the cell where they do their job) -- for instance, in a particular
organelle. Compartmentalization means that enzymes needed for
specific processes can be kept in the places where they act, ensuring
they can find their substrates readily, don't damage the cell, and
have the right microenvironment to work well.

For instance, digestive enzymes of the lysosome work best at a pH

around 5.05.05, point, 0, which is found in the acidic interior of the
lysosome (but not in the cytosol, which has a pH of about 7.27.27,
point, 2). Lysosomal enzymes have low activity at the pH of the
cytosol, which may serve as "insurance" for the cell: even if a
lysosome bursts and spills its enzymes, the enzymes will not begin
digesting the cell, because they will no longer have the right pH to
function.^55start superscript, 5, end superscript

Feedback inhibition of metabolic

pathways
In the process of feedback inhibition, the end product of a
metabolic pathway acts on the key enzyme regulating entry to that
pathway, keeping more of the end product from being produced.
This may seem odd – why would a molecule want to turn off its own
pathway? But it’s actually a clever way for the cell to make just the
right amount of the product. When there’s little of the product, the
enzyme will not be inhibited, and the pathway will go full steam
ahead to replenish the supply. When there’s lots of the product
sitting around, it will block the enzyme, preventing the production of
new product until the existing supply has been used up.

Diagram illustrating feedback inhibition. The end product of a multi-

step metabolic pathway binds to an allosteric site on the enzyme that
catalyzes the committed step of the pathway, reducing the enzyme's
activity. This regulation helps slow the pathway down when levels of
the end product are already high (when more is not needed).
Image credit: OpenStax Biology.

Typically, feedback inhibition acts at the first committed step of the

pathway, meaning the first step that’s effectively irreversible.
However, feedback inhibition can sometimes hit multiple points
along a pathway as well, particularly if the pathway has lots of
branch points. The pathway steps regulated by feedback inhibition
are often catalyzed by allosteric enzymes.^{6}6start superscript, 6,
end superscript
For example, the energy carrier molecule ATP is an allosteric
inhibitor of some of the enzymes involved in cellular respiration, a
process that makes ATP to power cellular reactions. When there is
lots of ATP, this feedback inhibition keeps more ATP from being
made. This is useful because ATP is an unstable molecule. If too
much ATP were made, much of it might go to waste, spontaneously
breaking back down into its components (ADP and P_iistart
subscript, i, end subscript).

ADP, on the other hand, serves as a positive allosteric regulator (an

allosteric activator) for some of the same enzymes that are inhibited
by ATP. For instance, ADP may act by binding to an enzyme and
changing its shape so that it becomes more active.^{7}7start
superscript, 7, end superscript

Thanks to this pattern of regulation, when ADP levels are high

compared to ATP levels, cellular respiration enzymes become very
active and will make more ATP through cellular respiration

Attribution:
This article is a modified derivative of “Enzymes,” by OpenStax
Biology (CC BY 3.0). Download the original article for free
at http://cnx.org/contents/185cbf87-c72e-48f5-b51e-
f14f21b5eabd@9.85:32/Biology.

The modified article is licensed under a CC BY-NC-SA 4.0 license.

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