SCHOOL OF MEDICAL
LABORATORY SCIENCE BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Kyle Vinci P. Solano RMT, MD
AY 2022 – 2023 - 1ST SEMESTER MODULE NO. 4
OUTLINE
I. Enzymes (Enzyme Kinetics)
A. Kinetics
II. Enzymes (Mechanism of Action and Clinical Enzymology)
I. ENZYMES (ENZYME KINETICS)
● Proteins
● Catalysts
→ Increase rate of chemical reaction exponentially
▪ Why do we need to increase the rate of chemical reaction?
Spontaneous reactions tend to occur however they are
slow. There are four ways to increase the chemical
reactions:
− (1) increasing the concentration of the reactants
− (2) increase heat
− (3) mechanical agitation
− (4) the use of catalysts
▪ For example: we have two molecules that bind together
forming a chemical reaction. When under the influence of a
solvent, the molecules will break off and these molecules
that are freely floating within the solvent will eventually
come in contact or directly interact with each other forming
chemical reactions. Under the influence or increase of
heat, there will also be an increase in kinetic energy thus
increasing the vibration of the atoms of the reactants. More
movement, more chances of direct interaction therefore a
greater possibility of interactions with these molecules
occur alongside numerous chemical reactions forming.
▪ The reactants, inherently, are 3-dimensional but not all
sides are optimal to have chemical reactions. Here comes
the catalysts that will allow these reactants' optimal sides to
face each other producing a chemical reaction. With the
use of catalysts, it bypassess the time when the molecules
are just freely floating waiting for it to bump with each other
to have chemical reactions
▪ With the use of catalysts, it increases the reaction rate up
to 10 times or more. It reduces the energy for molecules
going around looking for another reactant to bind with and
it reduces the energy and time a lot
▪ Enzymes are protein catalysts: produced by any biological
creature (can be produced by your bacteria, plants and
animals and it just so happens the viruses do not produce
it since these are only information and or data. In a context
that viruses need hosts in order to manufacture the needed
enzymes for them)
▪ Discovery of enzymes: when people are making bread,
they add yeast in turn these yeast will produce gas making
the bread rise and increase the chemical reactions. It
increases the rate of reaction exponentially by 10 but are
bound by the limitations of the proteins
● Bound by limitations of proteins
→ Proteins are resilient but they have factors that may inhibit
them
● Thermodynamic spontaneity cannot tell us whether a
reaction will be fast.
→ There are times when some reactions do not need the
assistance of heat and mechanical agitation to induce a
speedy chemical reaction.
● The speed of a reaction is a kinetic property controlled by the
nature of the energy state of the Enzyme - Substrate complex
and the transition state.
→ So what is the Enzyme-substrate complex?
BSMLS – 2G Team Writers: Kanashiro, Cataag
▪ The enzyme: has the active site meanwhile the substrate is
the reactant that goes into the active site. Once the reactant
will go to the active site, the chemical reaction will proceed.
Substrates could be in the form of nucleic acids,
carbohydrates, lipids, proteins and other enzymes. .
● Enzymes speed up the reaction rate by creating a situation
where the distance between the transition state and the ES
complex on an energy diagram is reduced.
● A chemical reaction may go faster at higher temperatures.
→ An increase in temperature there will be an increase in kinetic
energy
→ Remember that enzymes are also molecules and if subjected
to heat it will also move faster thus increasing the chances that
it will bind with a substrate.
→ Let us all remember that proteins are also prone to denaturing
when heat is applied. Per 1 degree of increase in temperature
there will be a subsequent ten times increase in enzymatic
activity. Eventually, it will get too hot, it will denature itself
leading to a zero in enzymatic activity (coagulation ensues).
● However, when the reaction is catalyzed by an enzyme, this is
true only for a specific range of temperatures.
● If the temperature is raised too much, it denatures the enzyme
and the rate of reaction is reduced significantly, perhaps to zero
Mode of Action
● Enzymes and substrates are attracted to each other via
noncovalent interactions, such as electrostatic attractions.
→ Why is it important for it to be non-covalent reactions? This is
because once the substrate is converted to its product it still
needs to dissociate from your enzyme. (NTK: covalent bonds
are much stronger than any type of bonds)
● The active site of an enzyme has amino acids in a specific
orientation where they can bind to the substrate
THE TWO MODELS OF BINDING .
● The Lock-and-key model is a description of the binding of a
substrate to an enzyme such that the active site and the substrate
exactly match each other in shape.
→ The substrate will bind perfectly with the active site. It means
the active site is rigid meaning it will not change
Figure 1. Lock and Key model
● The Induced-fit model is a description of substrate binding to an
enzyme such that the conformation of the enzyme changes to
accommodate the shape of the substrate
→ Means the enzyme’s active site can conform 2
● Note: The body is complex. There are times when some
molecules confer to have isomers or isoforms and scientists
concluded that the active sites of the induced fit model have the
ability to accommodate.
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LABORATORY SCIENCE BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Kyle Vinci P. Solano RMT, MD
AY 2022 – 2023 - 1ST SEMESTER MODULE NO. 4

Enzyme-catalyzed reaction

Figure 4. Enzyme-catalyzed reaction

Figure 2. Induced Fit
A. KINETICS
● In many situations the concentration of the reactants does
influence the rate of an enzyme-catalyzed reaction (First Order
Kinetics).
→ First order kinetics simply means that there is a rapid increase
in production of products during the initial increase of
substrate concentration. (rapid increase means exponential
increase)
● However, if there is very little enzyme and a saturating amount of
substrate, then all of the enzyme molecules are bound to
substrate.
● Adding additional substrate under this condition will not increase
the rate of reaction.
● When this happens, the enzyme is already working at its Vmax
and is exhibiting zero-order kinetics.
● Steady state is the condition in which the concentration of an
enzyme–substrate complex remains constant in spite of
continuous turnover.
Figure 3. The rate and the observed kinetics
● E: enzymes
● S: Substrate
● k1 or k-1: rate constant
● ⇋: reversible reaction
● ES: Enzyme-substrate complex
● P: product
→ If you want to reverse the product, you need another enzyme
Michaelis–Menten Equation
Figure 5. Michaelis-Menten equation
● V: velocity (it means the ½ vmax)
→ Michaelis constant (Km) a numerical value for the strength
of binding of a substrate to an enzyme. Which can be defined
most simply as the amount of substrate necessary to allow an
enzyme to function at half its maximal velocity.
▪ Simply means: it is substrate concentration that will give
you ½ vmax
▪ The purpose of michaelis constant: remember the antigen
in the body is the most specific reaction in all biology so we
can have the target. We just use an antibody to bind to the
enzymes. We tried to calculate the concentration of
enzymes using its substrate uptake
▪ If we use the Michaelis constant, we could also find the ½
vmax and if we multiply it by two we could derive our Vmax
thus our enzyme concentration
▪ Why is it important that we need to quantify the enzymes?:
Remember that if we have a lot of enzymes in our body
there may be damage to the tissue. In myocardial
infarction, there will be an increase in cardiac enzymes
CKMB or Creatine Kinase MB

- Velocity: refers to the rate of production

- Based from the graph, there is a plateau: the body of a
human can contain only so much enzymes so the plateau
position is described to be the rate limiting
- If the enzymes have a certain degree of concentration then
there will be a limit of a maximum velocity
- In first order kinetics: the enzymatic concentration is still
greater than your substrate (E>S).
- Zero-order kinetics: Eventually, the substrate → V, is equal to half the maximum rate possible, V = Vmax/2
concentration is greater than the enzyme (E<S). → Turnover number the number of moles of substrate that react
Dependent on enzyme concentration
per second per mole of enzyme
- Saturation: The active sites of the body are already full
→ k is a proportionality constant called the rate constant.
or occupied. It can't produce any product anymore

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Kyle Vinci P. Solano RMT, MD
AY 2022 – 2023 - 1ST SEMESTER MODULE NO. 4
● With only two substrates, we can already envision several ways
that the substrates and products can form complexes with the
enzyme.
● The most common mechanisms are called the ordered
mechanism, the random mechanism, and the ping-pong
mechanism.
→ Ordered mechanism: meaning the enzyme will have to bind to
the A first becoming EA then it binds to B again and then it
becomes AEB
→ Ordered mechanism: meaning the enzyme will have to bind to
the A first becoming EA then it binds to B again and then it
becomes AEB. When we look at the arrows, it signifies an
irreversible reaction.
→ (2) When we see reversible reactions it pertains to it being
random
→ (3) ping-pong mechanism will simply mean that E leads to EA
and will lead to a modification of E (that is why there is an
apostrophe on that E). The modified enzyme plus the product
and only the modified enzyme could bind to your B. After its
reaction with B it will return to its unmodified version that's why
it is called pingpong because it's pabalik-balik from modified
to unmodified.
Figure 6.
● Mathematically, KM is equal to the substrate concentration that
yields a velocity of Vmax/2.
● Mathematically, KM is equal to the substrate concentration that
yields a velocity of Vmax/2.
● It is also a crude measure of the affinity between the enzyme and
substrate, where a low KM indicates a high affinity.
● Vmax tells us how fast the enzyme can generate product under
saturating substrate conditions.
Figure 7. Graphical determination of Vmax and Km
BSMLS – 2G Team Writers: Kanashiro, Cataag
Lineweaver-Burk plot
● The Lineweaver–Burk plot is a double-reciprocal plot a graphical
method for analyzing the kinetics of enzyme-catalyzed reactions
● Comparing a Lineweaver–Burk plot of an uninhibited reaction to
one for an inhibited reaction, one can identify the inhibitor is
competitive if the curves intersect on the y-axis.
Figure 8. Lineweaver-Burk plot
● An inhibitor, as the name implies, is a substance that interferes
with the action of an enzyme and slows the rate of a reaction
● A reversible inhibitor can bind to the enzyme and subsequently
be released, leaving the enzyme in its original condition.
● An irreversible inhibitor reacts with the enzyme to produce a
protein that is not enzymatically active and from which the original
enzyme cannot be regenerated.
● Competitive inhibition a decrease in enzymatic activity caused
by binding of a substrate analogue to the active site.
→ It binds to the active site of the enzyme so the substrate could
not interact with the active site anymore. In order to reverse
this, we need to increase the concentration of substrates to
increase affinity. An increased affinity to the active site will
knock off the inhibitor.
● Noncompetitive inhibition is a form of enzyme inactivation in
which a substance binds to a place other than the active site but
distorts the active site so that the reaction is inhibited.
→ The inhibitor will bind to the allosteric site. Remember that
enzymes are proteins and there are parts of the proteins that
are highly interactive meaning that there is another binding
site. Allosteric site, simply means that it is a binding site that
is not an active site. An inhibitor binding to the allosteric site,
there will be a conformational change in the enzyme active site
● Uncompetitive inhibition is a type of inhibition where the
inhibitor can bind to ES, but not to free E.
→ This inhibitor could not bind to the active site directly and still
needs to wait until the substrate binds to the enzymes
therefore it will now bind to the complex
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LABORATORY SCIENCE BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Kyle Vinci P. Solano RMT, MD
AY 2022 – 2023 - 1ST SEMESTER MODULE NO. 4

your substrate comes along, a specific combination in your

substrate based on the charge will be attracted to the active
site, thereby, leading to a binding. This binding is not only quite
strong, but also quite specific. So after binding, the optimal
side (optimal 3d orientation of the substrate) for the chemical
reaction is also achieved, that’s why we’re getting a very fast,
and optimal chemical reaction. Usually it doesn’t have any
water because it tends to decrease the rate of reaction unless
water is needed (hydrolysis). Lots of things can bind there,
including your inhibitors in your non-competitive inhibition.

● (1) we just need to increase the substrate concentration. If ● Isoenzyme - refers to characteristically similar enzymes but with
you increase the substrate level to remove the inhibitors different genetic origins.
your km will also be increased but the Vmax, the rate of → If enzymes A and B do the same chemical reaction, and
product production is unaffected because the enzymes basically look the same, but differ in genetic origin.
are still alive and still active. Remember that Vmax is ● An Isoform results when an enzyme is subject to
dependent on your enzyme concentration posttranslational modifications
● (2) you have a reduced Vmax because once it is bound to → The same enzyme from the genetic level and from
the ES complex the competitive inhibitor can no longer be translational [origin]. After they are translated from your
used. The total enzyme population has decreased. ribosomes, they have an additional characteristic added, that’s
Decreased enzyme complex, decreased activity and why they’re quite different.
maximum saturation. ● In addition to the basic enzyme structure, a non-protein molecule,
● (3) the Vmax is reduced because the total enzymes is still called a cofactor, may be necessary for enzyme activity.
reduced → It aids, enables the enzymes to produce the chemical reaction.
● A suicide substrate is an irreversible inhibitor. It binds covalently They are also called INORGANIC MOLECULES and then we
to the enzyme inactivating it. Suicide substrates are important call them ACTIVATORS (Ions, Magnesium Chloride,
drugs in medicine and are used to study enzyme mechanisms. Selenium, Molybdenum).
→ If there are a lot of enzymes that are detrimental to the body. ● Inorganic cofactors, such as chloride or magnesium ions, are
Sometimes we need to use suicide substrate to stop those called activators. A coenzyme is an organic cofactor, such as
things nicotinamide adenine dinucleotide (NAD).
● When bound tightly to the enzyme, the coenzyme is called a
II. ENZYMES (MECHANISM OF ACTION AND prosthetic group. The enzyme portion (apoenzyme), with its
CLINICAL ENZYMOLOGY) respective coenzyme, forms a complete and active system, a
holoenzyme.
● As a protein, each enzyme contains a specific amino acid
sequence (primary structure), with the resultant polypeptide
chains twisting (secondary structure), which then folds (tertiary
structure) and results in structural cavities.
● If an enzyme contains more than one polypeptide unit, the
quaternary structure refers to the spatial relationships between
the subunits.
● Each enzyme contains an active site, often a water-free cavity,
where the substance on which the enzyme acts (the substrate)
interacts with particular charged amino acid residues
● An allosteric site - a cavity other than the active site - may bind
regulator molecules and, thereby, be significant to the basic
enzyme structure
→ So remember that enzymes are proteins and they have this
3D shape, and in this 3D shape, they usually make a cavity
and this cavity is lined by their amino acid residues. These
specific amino acid residues hold a different charge. So they
are making a specific combination based on the charge. When

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Kyle Vinci P. Solano RMT, MD
AY 2022 – 2023 - 1ST SEMESTER MODULE NO. 4

6. Ligases - Catalyze the joining of two substrates, coupled

VITAMIN with breaking of the pyrophosphate bond in adenosine
COENZYME REACTION TYPE
PRECURSORS triphosphate (ATP) or a similar compound

Biotin Carboxylation Biotin *Mnemonic: OTHLIL

Coenzyme A Acyl Transfer Pantothenic Acid

Oxidation -
Flavin Coenzymes Riboflavin (B2)
Reduction

Lipoic acid Acyl Transfer N/A

Nicotinamide
Oxidation -
Adenine Niacin
Reduction
Coenzymes

Pyridoxal
Transamination Pyridoxine (B6)
Phosphate

Tetrahydrofolic Transfer of One-

Folic Acid
Acid Carbon Units ENZYMES AND THEIR CLINICAL SIGNIFICANCE

Thiamine
Aldehyde Transfer Thiamine (B1)
pyrophosphate

● Some enzymes, mostly digestive enzymes, are originally

secreted from the organ of production in a structurally inactive
form, called a proenzyme or zymogen.
○ If zymogen is immediately activated, it kills the cell, all of the
proteins inside the cell, causing the cell to die.
○ In order to combat that dilemma, these types of proteins like
proteases are secreted as zymogens (inactive). In their amino
acid sequence there are additional and non-functional areas.
Because of the presence of the inactive amino acids, the
active site or the overall action of the enzymes is lacking. So
when it eventually goes to its target location (GI) which will
break down the protein intake, there is an enzyme that will cut
off.

Recommendations of the Nomenclature Committee of

International Union of Biochemistry and Molecular Biology on
the Nomenclature and Classification of Enzymes by the
Reactions they Catalyze
● Most enzymes are intracytoplasmic (intracellular), if there is
The -ase suffix from “diastase”, the first recognized enzyme. Its
tissue damage, that specific enzyme releases and causes an
usage in subsequently discovered enzymes was proposed by Emile
increase of amounts in the body. If we detect those, then we
Duclaux, with the intention of honoring the first scientists to isolate
can safely correlate them to the condition.
diastase.
● Ex. CKMB isoform is linked to myocardial infarction (heart
attack) = increase of enzyme is linked to the condition. The
1. Oxidoreductases - Catalyze and oxidation - reduction
only way for the enzymes to escape the intracellular place is
reaction between two substrates
for the tissue to undergo necrosis, or damage to the cell
2. Transferases - Catalyze the transfer of a group other than
(leaking cytoplasmic membrane)
hydrogen from one substrate to another
● Plasma concentration of these enzymes increases when
3. Hydrolases - Catalyze hydrolysis of various bonds
injury or malignancy of specific organs housing or secreting
4. Lyases - Catalyze removal of groups from substrates
without hydrolysis; the product contains them.
5. Isomerases - Catalyze the interconversion of geometric,
optical, or positional isomers

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Kyle Vinci P. Solano RMT, MD
AY 2022 – 2023 - 1ST SEMESTER MODULE NO. 4

acids, so they work well depending on the pH of the

solution)
4. Temperature - the rate of denaturation increases as the
temperature increases and is usually significant at 40°C to
50°C (directly proportional)
5. Cofactors/Coenzymes (no cofactor = cannot proceed
with the reaction)
6. Inhibitors

● Allosteric Site - another binding site within the enzyme that

is not the active site
● Allosteric Effectors - substances that bind to the allosteric
site that enter inhibitors (non-competitive inhibition),
substrates and activators.

Two Conformations of Enzymes:

● Active R (relaxed) conformation, which binds substrate
tightly
● Inactive T (tight/taut) conformation, which binds substrates
less lightly

THE NATURE OF THE ACTIVE SITE

● Only a few residues are directly involved in the active site,
but the whole molecule is necessary to provide the three-
dimensional arrangement for those critical residues
● Nucleophile - an electron-rich substance that tends to
react with sites of positive charge or polarization
● Electrophile - an electron-poor substance that tends to
react with sites of positive charge or polarization

FACTORS THAT INFLUENCE ENZYMATIC REACTIONS:

1. Substrate Concentration (First order kinetics)
2. Enzyme Concentration (Zero order kinetics - vmax)
3. pH - Enzymes are proteins that carry net molecular
charges. Changes in pH may denature an enzyme or
influence its ionic state, resulting in structural changes or
a change in the charge on an amino acid residue in the
active site (amino acid residues are dependent on the
binding of pH because of the acidic and the alkaline amino

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