ENZYMOLOGY In the presence of an enzyme in an enzyme catalyzed reaction, the energy of activation is much lower thus

by Dr. Bravo the energy barrier is easy to overcome, the reaction proceeds at a rate that requires lower energy making it
ENZYMES faster.
Special set of catalysts – PROTEINS The enzyme has to bind to the substrate in an area known as the active site.
From the Greek word énsymo meaning inside the yeast. o It should fit to the shape of the substrate.
Accelerate reaction rate within cells so that the reactions rates would be compatible for life.
Life would not be possible without enzymes.
Whatever factors affect proteins would also affect enzymes.
There are few that are made up of RNA called ribozymes, there are more protein enzymes.
Responsible for regulating metabolic reaction rate.
o If we want to speed up/slow down a particular reaction, all we have to do is control the enzyme.
Biologic catalyst
o Any other site on the enzyme other then the active site is known as the allosteric site.
HOW DO ENZYMES CATALYZE REACTIONS?
Any reaction would have a corresponding substrate/reactant and a product. THEORIES ON ENZYME-SUBSTRATE BINDING
For a reaction to be spontaneous, the free energy of the product should be less than the free energy of the Lock-and-Key Model
substrate. The enzyme’s active site fits the substrate perfectly.
o Spontaneous – it doesn’t mean it will proceed at a fast rate. When the enzyme approaches the substrate, there is
o The rate of the reaction will depend on the transition state – the energy requirement for the already an initial perfect fit between the substrate and the
transition state. enzyme’s active site.
 Transition state – the state between the substrate and the product that the substrate This explains why enzymes are specific when it comes to
has to undergo before becoming a product. substrate.
The energy difference between the transition state o If this enzyme is acting on a protein, any other
and the substrate is the energy of activation (∆G). molecule that isn’t a protein (e.g. Carbohydrate)
would not fit the active site and therefore
o Any reaction would have a corresponding would not be acted upon by the enzyme.
energy of activation. Limitation: implies that enzymes are very rigid, 1 enzyme: 1
substrate.
o The higher is the energy of activation, o This is far from the truth because enzymes are proteins and proteins are flexible molecules.
the greater is the energy requirement for o Although they are specific when it comes to substrate, they are not really that specific. As long as
the reaction to proceed. the substrates are interrelated to one another, enzymes can still act upon it.
Induced fit Model

E + S <---> [ES] <---> [EP] <---> E + P

In the presence of an enzyme (E), the temporary formation of the enzyme-substrate [ES] and eventually the
formation of the enzyme-product [EP] lowers the energy of activation.
o Enzymes catalyze reaction by lowering the energy of activation.
The enzyme should bind to the substrate.
o Any enzyme would have a unique shape that allows it to fit to the substrate or to the reactant.

Initially, there is no perfect fit between the active site and the substrate.
When the enzyme approaches the substrate, the substrate induces a change in the active site so
complementarity will be achieved – conformational change – eventually achieving a perfect fit.
Modification of the lock-and-key model.
Implies that the substrate can change the enzyme by inducing a conformational change in the active site.
Also implies that the enzymes can change the substrate by acting on it and turning it to a product.
This explains why enzymes act on a wide range of related substances.
o Example: Proteases
 Act on a variety of proteins.
 Proteins are interrelated to one another structurally.

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SIMPLE ENZYMES APOENZYME
Only made up of protein. Taken out cofactor or co-enzyme, only left with the protein portion.

CO-FACTORS
Small, inorganic, metal ions that are required of some enzymes to activate them.
Usually located near the active sit.
Helps in binding to the substrate.
Example: Mg++ stabilizes the carbonyl oxygen in the phosphoenol pyruvate to allow the enzyme enolase to
act on it; Mg++ participates in the catalytic activity.
PROSTHETIC GROUPS
There are certain cofactors or co-enzymes that are
difficult to separate from the protein portion of the
enzyme because they are covalently bound to the
enzyme.
Example: Heme
o The structure shown here that is
present not just in haemoglobin but
in other enzymes like catalase.
CO-ENZYME
Big, non-protein, organic molecules that are required of some enzymes to activate them. FACTORS THAT AFFECT ENZYME ACTIVITY
Participates in binding to the substrate. pH (power of hydrogen)
Binds to the active site to prepare the active site for substrate binding. o For most mammalian enzymes,
By them don’t have any catalytic activity. they work best at around pH of 7.
Help catalyze reactions by: o If we increase the pH, this results
o Donate/accept electrons in the inactivation of the enzyme
o Transfer group because the enzyme is denatured.
o Form/break covalent bonds However this is just the general
o Provide functional groups rule. There are a few enzymes
Common co-enzymes such as the ones in our stomach
o Lipoic acid (gastric enzymes) that work best
 Decarboxylate alpha-keto acid at around pH 3 and in the
o NAD/NADP, FAD intestinal enzymes work best at
 Redox reaction pH 10.
 Transfer of electrons
 Dehydrogenation Temperature
 Transfer of H+ o For most mammalian enzymes, they work best at 37oC.
o CoASH o If we isolate the enzyme and lower
 Kreb’s Cycle the temperature, this results in the
 Beta oxidation inactivation of the enzyme. The
o Vitamins enzyme is not destroyed; it can still
 The body needs vitamins such as ascorbic acid, cyanocobalamine and folic acid. be recovered if we slowly reach the
 By themselves, they do not provide energy but instead help unlock energy by acting as temperature back to 37oC.
co-enzymes for some metabolic reactions. o If we increase the temperature,
HOLOENZYME this usually increases the activity of
The entire enzyme together with all the necessary cofactor plus the protein portion. an enzyme up to a certain point.
The complete enzyme. There will come a point wherein
the activity of the enzyme is also
declining when increasing
temperature and eventually activity
will be lost or destroyed because
the enzyme has been denature
cannot be recovered anymore.
Substrate concentration

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HOW DO ENZYMES LOWER THE ENERGY OF ACTIVATION?  The usual requirement is that the active site of the enzyme should contain a polar
There has to be mechanisms employed by enzymes to help lower the energy of activation. amino acid that can act as a nucleophile. The substrate should also be polar; the typical
o Catalysis by alteration in proximity and orientation substrates are the phosphate groups, the acetate groups or the glycosyl groups, any
 Involved in bond formation. substrate that has a relatively polar center, like an electrophilic center.
 Say these are reactants in a solution
without any enzyme, the goal is to join
red and green molecules together, form
bonds between the 2 of them.
 Molecules in a solution should be
moving around form them to move
around so they can bump into each
other eventually forming a bond within each other.
 To ensure having a greater chance of bumping to each other, we must increase their
kinetic energy, keep them moving around and eventually form a bond.
 However, in the presence of an enzyme, the enzymes can bring those 2 molecules
together. This means less requirement for energy, they don’t have to move as much
around for them to collide to one another. Bond formation is possible at a lower
energy.
 In an uncatalyzed solution, say these
reactants bump into each other, but
there is another requirement that they
have to satisfy: they have to collide with
each other in the proper orientation, a
totally random process.
 In the presence of an enzyme, not only
are the substrates brought close to each other, they are also brought in the proper
orientation. That is why bond formation is not possible at a lesser energy requirement.
 This particular mechanism is true for enzymes that form bonds.  The picture given is the representation of the enzyme. X represents the polar amino
o Covalent catalysis acid. In this case, this has an outer electric pair on its surface and therefore it is
 Primarily involved in breaking bonds. negatively charged.
 This involves transient bonding between the amino acid residue in the active site and
that of the substrate.
 Generally, the active site of the enzyme should contain polar amino acids such as:
 Serine
 Threonine nucleophilic attack
 Cysteine
 Histidine
 Arginine  The usual substrates are the phosphate groups. So the goal here is to separate R from
 Lysine R’ (R prime). So we are going to cleave a bond.
 This is the overview of how the reaction is carried out:  Remember, the phosphorous (P) is partially positively charged because all the electron
clouds are moving towards oxygen (O). Since P is positively charged, the outer electron
pair on the enzyme will be attracted to it and would launch what is known as a
nucleophilic attack which results in the formation of a covalent bond resulting to the
 A and B are bound to each other. In the presence of water, there is separation of A and
breakage of the bond between R and R’.
B. Let us pretend X is the enzyme and X has a nucleophilic or polar group, the reaction
would be:
 If this has been A: then this would have been B: and they

 A and B are bound to each other in the presence of enzyme X, the X temporarily forms
a bond with A, this results in a cleavage of bond between A and B.
 The goal of separating A and B has been accomplished.
 In the presence of water, there is also breakage of the bond between A and X. There is
now separation from A and B.
have been separated from each other.
 In the presence of water, there is also cleavage of bond between the enzyme and
substrate. Thus, A and B have been separated from each other.

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o Acid-base catalysis  This reaction can be seen in glycolysis.
 Primarily involved in breaking bonds.  We call this metal catalysis because this involves metal cofactors. In this particular
 This is exemplified by chymotrypsin which is the enzyme secreted by the pancreas for case, the metal cofactor is Mg2+.
digesting protein. As a matter of fact, this is the mechanism employed by many
proteolytic enzymes, enzymes that digest protein.

 Mg2+ is positively charged and therefore it will be attracted to the O2. When it
approaches the O2, the O2 becomes even more negatively charged.
 This is a representation of a substrate. R1 is representation for amino acid 1; R2 is a  All the electron clouds migrate towards O2. This will render C here positively charged
representation for amino acid 2. They are joined together in what is known as a and therefore hungry for electrons.
peptide bond.  In the end, there is a partial negative charge in O and a partial positive charge in C
 The goal is to separate R1 from R2 by breaking the peptide bond. Chymotrypsin is the because of the attachment of Mg2+ which acts as the cofactor.
one that will accomplish that.  Now the enzyme enolase can act on it. Enolase, in its active site, will have a basic
 Chymotrypsin contains serine, an alcoholic amino acid. The functional group is OH- but amino acid like lysine. The basic amino acid contains an outer electron pair on its
it can also act as an acid. surface and therefore would be attracted to the H+ here which has lost most of its
 Another requirement is that the chymotrypsin, on its active site, should also contain a electron cloud to C.
basic amino acid like arginine and lysine.  The H+ will transfer resulting in the disruption in the electron cloud distribution and
 Why is this called acid-bas catalysis? Before an enzyme could act on substrate, there they would all migrate back.
has to be transfer of the H+ from serine to the base. This involves transfer of the H+ ion  The C will have an excess of electron. When there is excess of an electron here, the
from serine to the base. hydroxyl group will be cleaved off and would form a bond with H+ from glutamic acid
 The serine acts as an acid and the base which accepts the H+ acts as a base. and the end will be liberated in the form of HOH.
 When there is a transfer of H+ from OH- to the base, this in terms becomes hungry for  The final product is what is known as phosphoenolpyruvate, the dehydrated form of 2-
something that is positively charged. phosphoglycerate.
 The C is positively charged because all the electron clouds are moving towards O2.  The bottom line is that enolase would not have dehydrated this particular molecule
There is another nucleophilic attack. This results in bond formation. without the initial action of Mg2+ which acts as a metal cofactor hence metal catalysis.
 We have separated R2 from R1. In the presence of water, there will be another
cleavage of this particular bond, separating R1 from the enzymes. HOW ARE ENZYMES NAMED?
 It’s very similar to the first one except that before a nucleophilic attack can be There are 2 commonly utilized methods:
launched, there has to be transfer of H+. o Base the enzyme on the name of the substrate it acts on then add the suffix ‘-ase’.
o Metal catalysis  Substrate + ‘-ase’
 Primarily involved in breaking bonds.  Example: Urea + H2O → NH3
 This is exemplified by this reaction:  Urea in the presence of water would yield ammonia. The substrate is urea
and the enzyme would be called urease.
o More commonly, enzymes are named after their reaction that they do catalyze plus add the suffix
‘-ase’.
 Activity + ‘-ase’
 In this cartoon, the DNA is
being polymerized; a
phosphodiesther bond is being
formed.
 The reaction is called DNA
polymerization. Therefore we
can call the enzyme as DNA
polymerase.
 2-phosphoglycerate is dehydrated to phosphoenolpyruvate, which means that the OH-
and the H+ here have been cleaved off and liberated in the form of HOH (water).

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 o Another example:
 The name of the enzyme is pyruvate decarboxylase.
 The pyruvate here has been decarboxylated. The carboxyl group has been cleaved off
and liberated in the form of CO2.
o There are several exceptions to the given rule:
 Trivial names. Some enzymes, particularly proteolytic enzymes, they do not end
in the suffix ‘-ase’ rather end in ‘-sin’ like chymotrypsin and pepsin.
 Restriction endonucleases. These are enzymes that cleave DNA in a very specific
manner. They are named after the bacterial sources where they come from.
 EcoR1 – enzyme was taken from E. coli
 Mst2 – taken from an organism; M for the genus and st for the specie
HOW ARE ENZYMES CLASSIFFIED?
The International Union Biochemistry & Molecular Biology formed known as the Enzyme Commission.
This group divides a 4-didgit numbering system. Each particular known enzyme would bear, aside from its
official name, a 4-digit number.
ENZYME COMMISSION MAJOR CLASSES OF ENZYMES
Oxidoreductase
o As the name implies, they catalyze
redox reaction – transfer of electrons. One molecule
would lose an electron and another molecule would
eventually gain an electron. In organic chemistry, these
are also dehydrogenation reaction. As a matter of fact,
there is a subclass of oxidoreductase that is called
dehydrogenases like malic dehydrogenase, pyruvate
dehydrogenase, succinate dehydrogenase and alcohol
dehydrogenase.
Note:
When we are talking about oxidoreductase, the usual
coenzymes are NAD, FAD or NADP. Whenever we see an
enzyme that bears the name dehydrogenase, try to look
for the coenzyme right away. There should be NAD, FAD
or NADP in that particular reaction.
o Example: Alcohol dehydrogenase
o Alcohol/ethanol when taken in the
liver, alcohol is oxidized to yield acetaldehyde. It loses an
H+ as well as an electron. The electron is gained by NAD
so NAD has been reduced to NADH.

Transferases
o These enzymes catalyze the transfer of functional groups such as methyl, acetate and phosphate
among other things.
o There is a special class of transferases that are called kinases. They transfer phosphate usually
The first digit refers to the class of the enzyme. There 6 major classes of enzymes ranging from 1 to 6. coming from ATP.
The second digit refers to the subclass based on the type of bond acted upon by the enzyme. o One example is glucose. One of the very first reactions that glucose undergoes when it enters the
The third digit is a subclass within a subclass. cell is that it will be phosphorylated at C6. Phosphate will be attached C6.
The fourth digit is a serial number, the order in which that particular enzyme was added to the list. o Phosphate was transferred from ATP to glucose. From ATP it became ADP. The enzyme is called
Our concern is primarily on the first digit. hexokinase.
Hydrolyases
o They catalyze hydrolytic reactions, adding water across C-C bonds. These enzymes break bonds by
adding water molecule

o As we can see here in ATP, its bond is going to be broken and there is incorporation of water
molecule, which has been cleaved from each other.
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 Lyases secreted, they are usually cleaved to yield the active enzyme.
o Very similar to the enzyme hydrolyases are the enzymes lyases. They also break C-C bonds, C-O o Example: chymotrypsinogen
bonds, C-N bonds and other bonds. Sometime they generate a ring or a double bond. They break This is stored in the pancreas. Just before it is secreted, it is cleaved between arginine 15 (Arg 15)
bonds but do not incorporate any water and isoleucine 16 (Ile 16) to yield active chymotrypsin.
molecules. o Why is this advantageous way of storing proteolytic enzymes? When they are stored in their
o This reaction is catalyzed by pyruvate active form, the enzyme that stores them would eventually be digested and result to
decarboxylase. The bond between the 2 autodigestion without this particular mechanism.
Cs has been cleaved, making the carboxyl Allosteric regulation
group in the form of CO2. There is no o In this example, we can see
water molecule involved making a pathway. The initial
pyruvate decarboxylase a form of lyase. substrate A becomes B then
Isomerases becomes C and so on. The
o Also called mutases. final product is E. Each step
o They catalyze interconversion between is catalyzed by a respective
isomers. enzyme like A becomes B
o Isomers are molecules that have the same catalyzed by enzyme a.
structure but different configuration. o Feedback inhibition. If there is excessive
o Example: fumarate and maleate amount of the final product E, it can
In naming the product, they both can be provide feedback inhibition to the initial
interconverted. One of them can be the enzyme a to slow down the reaction.
product and the other the substrate or o How does the final product E inhibit the
vice versa. initial enzyme A? The usual mechanism is
Ligases allosteric regulation. Say we have the
o They catalyze condensation between 2 substrate initial enzyme A, it binds to the substrate
with splitting of ATP. at the site known as the active site but
o They form bonds between 2 molecules and there there is another site on the enzyme
has to be source of energy, and the usual source known as the allosteric site. The allosteric site can be a site for regulation. In this particular
of energy is ATP. example, if there is excessive amount of the final product E, the final product E can act as an
o One example is pyruvate carboxylase. Here, inhibitor molecule. It binds to the allosteric site
oxaloacetate is formed by condensation of then alters the conformation of the active site
bicarbonate and pyruvate. A bond here has been so that it can no longer bind to the substrate. In
formed and the source of energy is the splitting effect, that particular enzyme has been
of ATP to ADP in Pi (inorganic phosphate). inhibited.
o Ligate = to tie together o Precursor activation. Not all forms of allosteric
o Opposite of lyase. regulation are inhibitory in nature. Sometimes
they can activate an enzyme. This time, it is no
longer called an allosteric inhibitor but a
HOW ARE ENZYMES CONTROLLED/REGULATED?
positive modulator. In this example, we have a
 Enzymes are responsible for regulating metabolic less-active enzyme and a more-active enzyme.
reaction rates so there has to be mechanisms of
controlling these enzymes.
This would be the active site , and this
Anchoring enzymes in membranes
o The cell membrane is composed of 2 layers of
would be the allosteric site . In the
phospholipid and inserted into them are integral
presence of a positive modulator, the
proteins. Some of these integral proteins are
modulator binds to the allosteric site, changing
actually enzymes. When they are inserted into
the conformation of the active site so that it binds
membranes, it allows them to interact efficiently
to the substrate much better. In effect, it actually
with their substrate. They are locked into that
activates the enzyme.
particular area; having nowhere else to go
Covalent modification
therefore they can act efficiently with their
o One of the functional groups in the enzyme will be
substrate.
attached to another functional group. In this case,
Inactive precursors
phosphate was attached to serine, rendering the
o Proteolytic enzymes are stored as zymogens,
enzyme active. This form of covalent modification
inactive precursors. Once they have been
is what is known as phosphorylation. The source of phosphate is usually ATP.

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 o There are several forms of covalent modification depending on the type of functional group that they will not produce the enzyme lactase, which makes sense because lactase is not needed to
is attached to the enzyme. digest lactose. However, if we start introducing lactose into that particular media, they soon begin
synthesizing the enzyme lactase by expressing the lac operon.
o How do they sense that there is a need
o In the case of phosphorylation, the enzyme has been for the enzyme lactase? How do they
phosphorylated. sense that lactase is not needed? These
can be explained in terms of the lac
operon. The lac operon is composed of
o It is called adenylylation when an adenylate the lacZ gene plus other genes. The lacZ
has been added to an enzyme to alter its gene, which manufactures the enzyme
activity. lactase, is under the control of an
operator and a promoter.

o Uridylylation is when a uridylate has been

added to an enzyme.

o Say we grow the yeast without the sugar lactose. In the absence of lactose, there is protein known
as a repressor. The repressor binds to the operator. When it binds to the operator, the lacZ gene
o ADP-ribosylation is shutdown. The RNA polymerase that expresses the lacZ gene can bind to the promoter but it
cannot move forward because the road is blocked by the repressor. The RNA polymerase would
not be able to express the lacZ gene and therefore would dissociate, there would be no
production of the mRNA that would eventually produce the enzyme lactase. Which makes sense
because lactase is not needed because there is no lactose.

o Methylation, if we add a methyl group to the

enzyme. This, in effect, alters the activity of
the enzyme.

Regulation of enzyme synthesis

o The most sophisticated way of controlling enzymes.
o Genes are DNA which encode for proteins. Proteins are made through a process of transcription
and translation of a particular gene. So gene encodes proteins and enzymes are proteins.
o Some enzymes are said to be inducible which means the manufacturer of that enzyme can be
turned on or turned off. If there is a lot of the enzyme, then the manufacturer of that enzyme can
be turned off. If there is a need for that enzyme, then the manufacturer of that enzyme can be
turned on. As opposed to a constitutive enzyme, the enzyme is always there, whether it is needed o However, if we grow now the yeast in the presence of lactose, we now need the enzyme lactase.
or not. If this is the case, the repressor is there and the sugar lactose will now bind to the repressor. This
o An example of an inducible enzyme is the enzyme lactase which is encoded by the lacZ gene. LacZ changes the conformation of the repressor; it can no longer bind to the operator. The lacZ gene is
gene is the start of a group of clusters of genes known as the lac operon which can be turned on unlocked and can now be expressed. The repressor goes away; when the RNA polymerase binds
or turned off depending on the need of the enzyme lactase. to the promoter it can express the lacZ gene. There would be production of the mRNA which will
o The lac operon is usually found in E. coli or yeast. Yeasts do not ordinarily metabolize lactose. In be utilized for the production of the enzyme lactase that would digest lactose.
other words, they prefer glucose. If you grow them in a media without lactose and have glucose,

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APPLICATION OF ENZYMES INTRODUCTION TO ENZYME KINETICS
Medical Rate
o Diagnostics o Speed
 LDH and CK are used to monitor myocardial infarction, but nowadays non-enzymatic o Velocity
proteins are used like tropinin and myoglobin o Measure of change over a period of time
o Therapeutics Reaction rate
 Urokinase and streptokinase are used to lyse blood clot o Formation of products over a period of time
Research
o Thermostable DNA polymerase is used in PCR. PCR is the in vitro amplification of DNA, producing RELATIONSHIP BETWEEN RATE AND SUBSTRATE CONCENTRATION IN UNCATALYZED REACTION
billion copies of a particular DNA sequence which can be utilized for a procedure known as DNA
fingerprinting – portions of the DNA can be amplified and producing billion copies of the DNA
which can be electrophoresed in a gel and it will produce a banding pattern that will be unique for
that individual. Used for forensic and paternity purposes.
Industrial
o There are certain genes that are subjected to enzymatic digestion to render them soft and
luxurious.

Questions:

1. At about 0 C., most enzymes are – inactive

2. At high temperatures, the rate of enzyme action decreases because the increased heat – alters the
active site of the enzyme
3. Vitamins are essential to the survival of organisms because vitamins usually function as – coenzymes
4. A certain enzyme will hydrolyze albumin but not starch. Which statement best explains this
observation? – Enzyme molecules are specific in their actions. Zero order Reaction First order Reaction
5. Which statement best expresses the information represented in the graph shown? Even if we increase the reactant concentration, Even if we increase the reactant concentration,
the reaction rate stays the same, it doesn’t there is a corresponding increase in the
change, it is constant. reaction rate.
The reaction rate is independent on the Direct, proportional relationship between the
reactant concentration. Even if there are a lot reactant concentration and the reaction rate.
of reactants, the reaction rate remains The reaction rate is dependent on the reactant
constant. concentration. The more the reactants, the
faster the reaction rate.
- The action of enzymes varies with pH.
6. Which statement best describes the enzyme represented in the graphs below? These 2 graphs don’t describe enzyme-catalyzed reactions, it’s neither a flat, straight line nor a flat ascending
line, and it’s rather a hyperbolic curve called the Michaelis-Menten curve.

MICHAELIS-MENTEN CURVE
We plot the reactant or the substrate concentration against the reaction rate. What is obtained is a
hyperbolic curve. It is a combination of the zero order and the first order.

- This enzyme works best at a temperature of 35 C and a pH of 8.

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In the first part of the process, when the substrate concentration When the Km is very high, it takes a lot of the substrate for the reaction to reach Vmax, which means the
is very low, it’s almost like in first order. If we increase the enzyme is not easily saturated. High Km means low substrate affinity. We always take a look at the Km to
substrate concentration, there’s a corresponding increase in the determine the enzyme’s affinity to the substrate.
reaction rate, hence first order. However, when the substrate Brain Exercise: Glucokinase and hexokinase are
concentration is very high, it now assumes zero order kinetics. No transferases. They transfer phosphate to glucose. One
matter how much substrate we add, it remains flat, it doesn’t of the very first reactions that glucose will undergo
change, it remains constant – zero order. when it enters the cell is that it will be phosphorylated
Somewhere in between, there’s a transition from first order to zero order, this is called mixed order. at C6 to yield glcose-6-phosphate. There are said to be
The Michaelis-Menten curve is actually plotting substrate concentration versus the reaction velocity. isozymes – they catalyze same types of reaction but in
Why is it that when the substrate concentration is low, it follows different organs of the body. One of them is located in
first order kinetics? Whereas when the substrate concentration is the liver and the other one is located in the muscle.
very high, it follows zero order kinetics? This is due to the o Between the 2, who has the higher Km? Who has the lower Km?
saturation of the enzyme. When the substrate concentration is  Glucokinase has a higher Km, lower affinity to the substrate.
very low, there are more enzymes than there are substrates, if we o What is the importance of their substrate affinity with respect to their organ location?
add more substrate, naturally more enzymes would be working  Glucokinase would only phosphorylate glucose when there is an abundance of glucose
and therefore there’s a corresponding increase in the reaction so the glucose in the liver can be stored. Hexokinase located in the muscle, on the
rate. The reaction rate would concurrently increase as well with other hand, needs to have a high affinity for glucose because the muscle is energy
subsequent addition of substrate. hungry.
But if we keep on adding more substrate, there will come a point where all the enzymes are already working. The Michaelis-Menten curve can be described using the following formula:
We have reached a point wherein no manner how much substrate we add, there’s no change in the reaction
rate, because the enzyme is said to be saturated. The reaction rate has reached what is known as Vmax, the
maximum velocity of reaction. No matter how much substrate is added, the Vmax won’t change anymore.

Halfway through the Vmax, it is called the V0 (reaction velocity) is equal to Vmax multiplied by [S] (Substrate concentration) over Km plus [S]. It is non-
Vmax ½. This corresponds to a substrate linear relationship. This formula describes the relationship between reaction velocity of enzyme-catalyzed
concentration. The substrate concentration reaction and substrate concentration.
and half of the Vmax is what is known as Km
(Michaelis constant). This tells us something HOW TO CHANGE MICHAELIS-MENTEN TO LINEWEAVER-BURKE EQUATION
about the enzyme’s affinity to the substrate. We try to convert the Michaelis-Menten curve into a linear formula so that we can readily derive Vmax and
Km.

In this particular example, there are 2

curves – the same Vmax but different
Km. One has lower Km and the other
one has a higher Km.
Between the 2, which one do you think
has a higher affinity for the substrate?
Which one has the lower affinity for the
substrate? When the Km is very low, the
Vmax is readily reached, even in a low
substrate concentration because the
enzyme is saturated easily.
Low Km means high affinity for
substrate. The reverse would be true.

9 KRISTINA PATRICIA L. ADALEM, RN

FEU-NRMF MEDICINE
 ENZYME INHIBITION
We study the Vmax, Km, Lineweaver-Burke and the Michaelis-Menten because we can use this to determine
the different types of enzyme inhibition.

Competitive inhibition. The substrate and the inhibitor

compete for the same binding site which is the active
site. If it’s the substrate that binds to the enzyme, well
and good. If it’s the inhibitor that binds to the enzyme,
this turns the enzyme inactive. This is a reversible type
of inhibition. The inhibitors often resemble the substrate
structurally; they are said to molecular analogues.

How do we make sure that the substrate is the one that

will bind to the active site? We increase the substrate
concentration. More substrate means that there is
greater likelihood that it’s the substrate that will bind to
the active site rather than the inhibitor.

What we plot now is 1/[S] versus 1/V0. 1/V0 is the y, the x is 1/[S], 1/Vmax is the y-intercept and if we extend We can still reach the Vmax as long as we have a lot of
all the way to the negative side, the x-intercept is the negative of 1/Km. substrate. The Vmax doesn’t change.
We have 2 curves here: the blue one is without the
For example, we want to determine Vmax and Km, this is the data that is given – substrate concentration inhibitor, the gray one is in the presence of the
with the corresponding reaction rate with reaction velocity. We will try to plot this data S versus V competitive inhibitor. The substrate can compete
(Michaelis-Menten). For us to against the inhibitor.
say that this is a Vmax, there Notice that the Km has been reset to a higher value. So
should be flattening, if we in competitive inhibition, the Km increases but the Vmax
continue adding more doesn’t change.
substrate, they should not
change anymore. But if we
keep on adding more substrate This is what it looks in Lineweaver-Burke. The green
and it keeps on rising, it not yet line represent the line without the inhibitor, red is in
the Vmax. We have to convert the presence of the inhibitor. Notice that they have
this to the Lineweaver-Burke, the same y-intercept which means 1/Vmax is the same
plotting it the reciprocal of which ultimately means that the Vmax has not
Michaelis-Menten. In x it would changed.
become 1/[S] and in y it would
become 1/V0. Plotting this
would obtain a linear graph.

Extrapolating this all the way

to the negative side, we can Non-competitive inhibition. They are not competing with
now derive Vmax from the same binding site. The substrate binds to the active site,
1/Vmax, also derive Km from the inhibitor binds to another site called the allosteric site.
1/Km. The inhibitor can bind to the free enzyme, it can also bind to
the enzyme that is already bound to the substrate. Inhibitors
never bind to the active site. Both the EI and EIS complexes
are enzymatically inactive.

10 KRISTINA PATRICIA L. ADALEM, RN

FEU-NRMF MEDICINE
 Mixed inhibition. In terms of mechanism, it resembles
non-competitive inhibition. The substrate and
Even if we add a lot of substrate, it cannot overcome the inhibitor have different binding sites. Inhibitor binds to
inhibitor. The original Vmax cannot be achieved anymore even the allosteric site, substrate binds to the active site.
if we add a lot of substrate, it has decreased. The Km doesn’t The inhibitor can bind to the free enzyme or to the ES
change, it remains the same. complex. There’s a difference, however, it’s not
complete inhibition, there is still what we call residual
activity – the enzyme is still somewhat working so
product can still be formed but at a much lower rate.
Inhibitors often act as feedback inhibitors. Enzymes
are often multimeric – made up of several subunits.

In the presence of the inhibitor, the Vmax decreased

In the Lineweaver-Burke graph, green is without
and the Km Increases.
the inhibitor and red is with the inhibitor. Notice
that they have the same x-intercept, which is
Km Vmax
1/Km. Ultimately means that Km is the same in
the presence of the inhibitor. Competitive inhibition Increases No change
Non-competitive inhibition No change Decreases
Uncompetitive inhibition Decreases Decreases
Mixed inhibition Increases Decreases

Uncompetitive inhibition. The inhibitor does not bind to DIXON PLOT

the free enzyme, it only binds to the enzyme once the How do we quantify potency of an enzyme inhibitor? We use the Dixon plot, it’s 1/v on the y-axis versus the
enzyme is already bound to the substrate. It binds to an concentration of the inhibitor at the x-axis. So it’s [I] versus 1/v.
allosteric site, substrate first followed by the inhibitor. This is performed at varying concentrations of the substrate. This will give us what is known as Ki (inhibitor
Inhibitor binds only to the ES complex. EIS complex is constant), this is an indicator for inhibitor potency which means the lower is the Ki, the more potent is the
inactive. Rare but may occur in multimeric proteins. inhibitor. Ki is the concentration of the inhibitor required to produce half maximum inhibition.
Lower Km means higher enzyme affinity, inhibitor is more potent.
For a competitive inhibitor, we plot the concentration of an inhibitor versus 1/v or the reciprocal of the
reaction velocity. This is done at varying substrate concentration. We obtain a family of intersecting lines, at
the point where they will intersect, this corresponds to the Ki. The smaller is the absolute value means the
Say that the purple line is wthout the inhbitor, the orange is in the presence of an inhibitor. The Vmax has inhibitor is more potent.
decreased, Km deacresed. In non-competitive inhibition, it may seem like it’s a variation of the Lineweaver-Burke except that in
Lineweaver-Burke, it’s 1/*S+ versus 1/V0 but here it’s inhibitor concentration versus 1/v.

11 KRISTINA PATRICIA L. ADALEM, RN

FEU-NRMF MEDICINE