[TRANS] LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

ENZYMOLOGY
• Tertiary
• Study of enzymes which involves:
o Secondary structures that
o Activity of enzymes are oriented in a three-
o Chemical reaction it catalyzes dimensional manner
o Clinical use
▪ Clinical enzymology
• Quaternary
 tackles about enzymes of clinical significance
o Combination of the different
 E.g. amylase, lipase, transferases, and other subunits of tertiary structure
miscellaneous enzymes
• Term “enzymology’” – for biochemistry • Some of the enzymes are
actually composed of more than one subunit; while
others only have one
ENZYMES
• Proteins that are composed of amino acids How the Enzyme Works
o Amino acids are composed of:
▪ a central carbon
▪ a functional group (depends
on the type of amino acid)
▪ carboxylic acid group
▪ amino group
o Polypeptide – chain of amino acids
o Peptide bonds – join the amino acids together
o Proteins have different functions:
▪ E.g. structural or catalysts
• Biologic catalysts
• Hasten chemical reactions
o Chemical reactions in the body may take a long time • The enzyme works by lowering the energy barrier for a
to proceed to a reaction. Hence, enzymes speed up reaction to occur.
the process up to a nanosecond. • Graph:
• Not consumed during the reaction o Y axis: Activation energy used for the reaction
o In an enzymatic reaction, the substrate becomes the o X axis: Direction of the reaction
product; whereas the enzyme remains the same. ▪ Going to the right = the reaction occurs
• Does not undergo chemical change after the reaction • Dotted lines:
o (1) Before the reaction occurs, there should be an
4 Domains of Structure of Proteins energy input in the system.
• Primary ▪ ANALOGY: The paper breaks into several
pieces because there is an energy introduced in
o The structure of the the system that will tear apart the paper.
enzyme on an amino
acid by amino acid level o (2) The energy barrier in the presence of an enzyme
o The amino acid becomes low.
sequence present in a
▪ The activation energy needed for the reaction to
particular enzyme
occur decreases in the presence of an enzyme.
• Secondary  Thus, the reaction becomes easier.
o How polypeptides are ▪ ANALOGY: The paper can break itself into
twisting pieces given enough time, but it can be easily
o E.g. alpha helix & beta breakdown into several pieces with the help of
pleated sheet the hand (enzymes).

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 1
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

Enzyme Location Nomenclature of Enzymes

• Found in all body tissues • 3 ways of naming enzymes:

o The human body is a walking chemical laboratory o Substrate + -ase
wherein each of the cell has its own chemical o Reaction it catalyzes
laboratory. o Enzyme Commission (E.C.) Nomenclature
o Since there are several reactions that need to occur
for a cell to live, there should be enzymes. Substrate + -ase

• Appear in serum/plasma: • Depending on the substrate upon which the enzyme

o Following cellular injury acts upon
• Examples of substate and its corresponding enzyme:
▪ Enzymes appear in an abnormal or
o Lipid = Lipase
pathophysiological circumstance.
o Ester = Esterase
▪ Example 1: Creatinine Kinase – MB (CK-MB)
 Specific to the cardiac muscle tissue ▪ Substrate: molecules that contain ester bond
 Found inside the muscular cardiac muscle o Protein = Protease
tissue of the heart o Starch = Amylase
 A heart injury such as acute myocardial ▪ Named as such because the two components of
infarction (heart attack) can cause damages starch are amylose and amylopectin.
to the cardiac muscle tissue which releases
CK-MB in the bloodstream. Reaction it catalyzes
 The presence of an increased CK-MB in the • Oxidase
serum or plasma means that there is an injury
to the tissue where the enzyme is found. o Oxidation – Removing of electron/s from a molecule
▪ Example 2: Lactate Dehydrogenase (LDH) • Reductase
 Since the LDH is found inside the RBCS, an o Reduction – Addition of electron
increased LDH may indicate intravascular or
extravascular hemolysis. • Hydrolase
o In smaller amounts, degraded cells o Hydrolysis – Addition of water to breakdown a
o For physiologic processes, like coagulation molecule

▪ Enzymes are needed in the serum or plasma • Dehydrogenase

such as in the case of coagulation. o Removal of H+, transferring them to a coenzyme
Two Sites of Enzyme • Decarboxylase
Active Site o Removal of Carboxyl groups

• Where the substance on which the enzyme acts • Deaminase

(substrate) or interacts with particular charged amino o Removal of Amino groups
residue.
• The site where the substrate will fit into to undergo an Enzyme Commission (E.C.) Nomenclature
enzymatic reaction for it to become a product.
• Standard process for naming enzymes
• It is the sub-department of International Union for
Biochemistry (IUB)
• 4 types:
o Numerical code
▪ E.C. 3.2.1.1. (for amylase)
▪ 1st digit = class
▪ 2nd digit = subclass
▪ 3rd and 4th = sub subclass & serial number
Allosteric Site o Systematic name (full name)
• Cavity other than the active site ▪ 1,4-D-Glucan Glucanohydrolase (for amylase)
• May bind regulator molecules o Recommended name (nickname)
o Examples: Co-enzymes, Activators ▪ α-Amylase (for amylase)
o Most of the enzymatic reactions need another
regulator molecule before it undergoes enzymatic o Abbreviated name
reaction. ▪ AMS (for amylase)

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 2
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

CLASSIFICATION OF ENZYMES • Forward Reaction

• (1) Oxidoreductase o If lactate is more than the pyruvate, the reaction will
be forward reaction
• (2) Transferase
o Lactate → Pyruvate
• (3) Hydrolase
• (4) Lyases ▪ Lactate = substrate
• (5) Isomerase ▪ Pyruvate = product
• (6) Ligases o Substrate is always in the name of the enzyme
• The number/order corresponds to the 1st digit of the
numerical code. • Reverse Reaction
o If pyruvate is at large amounts in a test tube, lactate
MNEMONIC: Oh To Hold Lyka’s Incredible Legs dehydrogenase turns it to lactate
o Lyka = Lyase o Pyruvate → Lactate
o Legs = Ligase
▪ Pyruvate = substrate
(1) Oxidoreductase ▪ Lactate = product
Oxidoreductase Reaction
• Oxidation and reduction
o The transfer of H+ ion from one substrate to another
to form the products
▪ Oxidation – removal of H+ ion
▪ Reduction – acceptance of H+
• Example: E.C.1.1.1.27
o Systematic name: L-Lactate NAD+
Oxidoreductase
▪ NAD – nicotinamide adenine dinucleotide • Dehydrogenase – One hydrogen is removed thus
becoming pyruvate
 NAD+ – oxidized form of NAD
o The oxidized NAD will receive the removed
 NADH – reduced form of NAD; due to
hydrogen and become reduced.
acceptance of hydrogen
o Recommended name: Lactate Dehydrogenase • In every oxidation-reduction reaction, one of the
o Abbreviated name: LDH substrates is oxidized to form the pyruvate while the
other is reduced.
Anatomy of the Enzymatic Reaction Symbols o Oxidation-reduction is always a pair
▪ Substates: Lactate, oxidized NAD (NAD+)
 Lactate is oxidized since H+ ion was
removed.
 Oxidized NAD (NAD+) becomes reduced
NAD (NADH) since it received the H+ ion
• Substrate NOTE: Whenever an enzyme ends in dehydrogenase, its
o Always at the left side function is oxidation.

• Product o Lactate dehydrogenase oxidizes lactate.

o At the right side (2) Transferases

• Arrow • Has a similar function to oxidoreductase
o Indicates the direction of the reaction • Transfer of functional groups other than hydrogen from
o Substrate may be on the right if the arrow is one substrate to another
reversed • Example: E.C. 2.6.1.1.
o Double arrow o Systematic name: L- Aspartate: 2-Oxaloglutarate
▪ Indicates that the enzymatic reaction is Aminotransferase
reversible ▪ Substrates for the forward reaction:
▪ “reversible” – can go either way
▪ Lactate → Pyruvate  L-aspartate, 2-Oxaloglutarate
▪ Amino group is being transferred thus the name
o The direction of the reaction depends on the amount
aminotransferase
of either the product or the substrate
o Recommended name: Aspartate Aminotransferase
• Always put the name of the enzyme above the arrow o Abbreviated name: AST
indicating that the enzyme is not changing after the
reaction ▪ Old name: SGOT

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 3
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

Transferase Reaction o Adding water will break the bond and turn amylose
into:
• Example: E.C. 2.6.1.2. ▪ glucose (monosaccharide) or
o Systematic name: L-Alanine: 2-Oxaloglutarate ▪ maltose (disaccharide) or
Aminotransferase ▪ dextrins (oligosaccharides)
o Recommended name: Alanine transaminase • Amylose can break by itself by adding water but
o Abbreviated name: ALT amylase can speed up the reaction
o Old name: Serum Glutamate Pyruvate • Amylase is found in the saliva (salivary glands) and
Transaminase (SGPT) pancreatic secretions (pancreas)
▪ Contains the name of the products of the o Ex. Rice (starchy) has amylose
reaction
▪ α-1,4 glycosidic bonds will break into
• α-ketoglutarate glucose/maltose/dextrins with the aid of water
o Another name for Oxaloglutarate and amylase
o Pancreatic amylase – further degradation of
carbohydrates
(4) Lyases

• Addition of a group to a double bond to become a single

bond, or
• Removal of a group to form a double bond
• Example: Carbonic Anhydrase & Citrate Lyase
Lyase Reaction

• ALT is an aminotransferase
o The amino group from L-Alanine is transferred to α-
ketoglutarate
▪ L-Alanine becomes pyruvate
▪ α-ketoglutarate becomes L-Glutamate
 An α-ketoglutarate that gained an amino • Carbon dioxide and water becomes carbonic acid with
group the help of carbonic anhydrase
 Transfer of amino group o Lyase – adds something to a double bond (water)
and becomes a single bond
• It is a forward reaction
o Substrate is in the name of the enzyme. • Blood pH
o Carbon dioxide in the lungs/bloodstream reacts with
(3) Hydrolases water with the help of carbonic anhydrase producing
• Hydrolysis of various bonds carbonic acid which becomes bicarbonate

o Addition of H2O to a bond resulting in bond ▪ Bicarbonate acts as a buffer for blood pH
breakage (5) Isomerases
• Example: E.C. 3.2.1.1.
• Rearrange the functional groups within the molecule
o Systematic name: 1,4-D-Glucan and catalyze the conversion of one isomer into another
Glucanohydrolase
o Isomers are the same molecules (same number of
▪ D – stereoisomerism atoms) but with a different arrangement
o Recommended name: α-Amylase • Example: Phosphoglycerate mutase
▪ Only acts on α-1,4 glycosidic bonds o Mutase – an isomerase; “mutates” a molecule into
another type of molecule
Hydrolase Reaction
Isomerase Reaction

• Amylose has a glycosidic bond (α-1,4 glycosidic bond)

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 4
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

o Has 3 isoenzymes, which are composed of 2

• 3-Phosphoglycerate
subunits of the tertiary structure of a protein:
o Glycerate (3 carbons) but the phosphate group is in
the 3rd carbon ▪ CK-BB – B and B subunits (migrate fastest
o Phosphoglycerate mutase will transfer the towards the anode/ travels the farthest)
phosphate to the 2nd carbon and becomes 2- ▪ CK-MB – M and B subunits
Phosphoglycerate ▪ CK-MM – M and M subunits (slowest mobility)
o Same function but differ in the subunits of their
(6) Ligases proteins
• Catalyze a reaction in which C-C, C-S, C-O or C-N • Isoenzymes have different physical properties which
bond is made or broken includes electrophoretic mobility, solubility and
o Bond formation or breaking down of a bond (if resistance to activation.
reversible reaction) o If undergo electrophoresis, they have different
▪ Energy – needed to form a bond mobilities.

• Accompanied by an ATP-ADP conversion or a similar ▪ Electrophoresis – serum is added in the agarose

compound such as GTP (nucleotide triphosphate) gel,  electric current is introduced for it to flow
from cathode to anode.
Ligase Reaction
 They have different speeds at which they
travel the agarose gel.
o Some are easily activated while some are not easily
activated.

Isoenzyme vs. Isoforms

• Central Dogma of Life

𝑡𝑟𝑎𝑛𝑠𝑐𝑟𝑖𝑝𝑡𝑖𝑜𝑛 𝑡𝑟𝑎𝑛𝑠𝑙𝑎𝑡𝑖𝑜𝑛
𝐷𝑁𝐴 (𝑟𝑒𝑝𝑙𝑖𝑐𝑎𝑡𝑖𝑜𝑛) → 𝑅𝑁𝐴 → 𝑃𝑟𝑜𝑡𝑒𝑖𝑛

• Enzymes are proteins

• DNA Ligase
o Embedded in the genome in the same way that
o Whenever there is a DNA strand in the 3’ end, body produce them.
another DNA should be added. o Genome has genes that encode for an enzyme
o If the triphosphate will break (2 phosphates will be
removed) with the help of ATP → Monophosphate • Isoenzyme
will create a phosphodiester bond o Structurally, it has different enzymes because they
• DNA Polymerase arise from different genes.
o Different genes which encode for different proteins
o When dinucleotide is added, it is accompanied with
ATP-ADP conversion. • Isoforms

NOTE: No ligase in the clinically significant enzymes so far. o Same genes and same proteins after translation but
differ during the post-translational modifications in
the Golgi complex.
TERMINOLOGIES o Different in structure because they were modified by
Substrate the Golgi complex differently.
▪ Ribosomes – where proteins are translated
• Molecule acted upon by enzyme
▪ Golgi body or complex – process the protein
• Specific for every enzyme produced from translation
• E.g., Amylase
 Also called as post-translational processing
o Substrate: α-1,4-glycosidic bond between amylose
and amylopectin Cofactor
o If triglyceride is introduced to amylase, nothing will
happen since that enzyme is specific for α-1,4- • May be necessary for enzyme activity
glycosidic bond. • Non-protein molecule
o Helps in the enzyme activity
Isoenzyme
o It goes into and connects to the allosteric site
• Iso – “the same”
Two types of Cofactors
• Same enzymes with the same action but have different
forms • Activator
• E.g., Creatine kinase
o Inorganic cofactor
o Kinase – involves ATP-ADP, but these are not o E.g. Calcium, Magnesium, Manganese, Zinc, Iron
ligases.

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 5
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

• Coenzyme Example 3
o Non-protein
o Organic cofactor
o E.g. Adenine Triphosphate (ATP), Nicotinamide
adenine dinucleotide (NADH)
▪ The larger the molecule, the more organic it is.
o Prosthetic group
• Apoenzyme + substrate + absence of cofactor
▪ When coenzyme is bound tightly to an enzyme
▪ Already present in the enzyme, not a separate o No enzyme-substrate complex, no reaction
entity from the enzyme o Needs a cofactor
Holoenzyme

• Apoenzyme + Coenzyme
o Absence of cofactor = no reaction

• Apoenzyme + Substrate + presence of cofactor =

reaction occurs
o Cofactor: Copper
▪ Activator (Inorganic element)
Proenzyme
Apoenzyme • Also known as zymogen
• Polypeptide portion of the enzyme o Generates the actual enzyme
o Protein portion of the enzyme that already contains ▪ Zymogen = “gen” - genesis
tightly bound coenzyme
• Inactive form of enzyme
Example 1
o Example: Preproprotein, undergoes post relational
modification in the Golgi complex and forms
proprotein, to protein
▪ Preproprotein → proprotein → protein
▪ It has to be converted first into an actual enzyme
before activated
o Where is it activated?
• Nicotinamide adenine dinucleotide (NAD) ▪ Depends on the enzyme
▪ Inactive form of enzyme reaches its destination
o Considered a substrate and as a coenzyme
before activated, usually by cutting amino acid
▪ Coenzyme – Organic cofactor because lactate • It is then converted, usually by proteolysis, to the active
dehydrogenase cannot proceed with the reaction form when it has reached the site of its activity
without NAD.
Example 2 ENZYME KINETICS
• How does enzyme behave inside the body or in the test
tube (laboratory)
• Kinetics “to move”

• Adenosine triphosphate (ATP) Michaelis – Menten Theory

o Organic cofactor • Leonor Michaelis (1875-
▪ Thus, it is a coenzyme 1949) and Maud Menten
(1879-1960)
o Involved in Creatine Kinase • Hypothesize the role of
substrate concentration in the
formation of the ES Complex

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 6
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

• Basic tenet of enzyme kinetics Bond Specificity

• E + S → E-S → P + E
• The enzyme is specific to a particular bond.
o An enzyme-substrate complex must first be formed
for an enzymatic reaction to occur o E.g., Proteases
▪ The product can only be achieved, if the ▪ Proteases digest proteins because they are
substrate forms a complex with the enzyme. particular to a peptide bond.
▪ There must be a physical interaction between ▪ Regardless of the type of amino acid, as long as
the enzyme and the substrate they are connected by a peptide bond.

Stereoisometric Specificity

• The enzyme will only act upon a specific isomer of

substrate.
• Base on arrangement of molecules
• (1) At first, there is a Substrate [S] and an enzyme [E] o E.g., Glucose dehydrogenase
• (2) Before the Substrate [S] turns into a Product [P],
there must be a formation of ES complex [ES] ▪ Classification of enzyme: Oxidoreductase

o ES Complex [ES] - the physical binding of the  “dehydrogenase” – removal of hydrogen

substrate to the active site of the enzyme ▪ A stereoisometric specific enzyme because it
only acts upon β-D-glucose.
▪ Crucial intermediate step in the enzymatic
reaction  If the glucose is an α-D-glucose, the glucose
▪ Explains the behavior of enzyme dehydrogenase will not react only to β-D-
glucose.
Analogy on Enzymatic Reaction of the Body

• How the enzyme work?

o If there are substrate and an enzyme, the enzyme
and substrate must form a complex. Then the
enzyme will turn the substrate into a product
• The enzymatic reaction is dependent on the availability
of the active site.
o The active site must be available because the
substrate must be place on the active site before the
• Enzyme is still the same enzyme at the end of reaction product is formed.
o Enzyme does not change chemically after the
reaction
Specificity

Absolute Specificity

• The enzyme can only form a complex to a specific

substrate.
o E.g., Amylase • The graph above shows constant amount of enzymes
▪ Amylase can only react with amylose because o Increasing the amount of substrate will increase the
amylase has absolute specificity towards speed of the reaction. However, it will eventually hit
amylose. a limit (plateau) despite adding more substrate since
▪ If alanine, albumin, and triglyceride are added, the active site is limited.
amylase will not react to it. o Enzymes cannot cater adding more substrate
Group Specificity • Adding more substrate in any enzymatic reaction will
lead to a limit or maximum velocity.
• The enzyme is specific for a particular functional
group. o X axis
• Regardless of the molecule, for as long as there is a ▪ Substrate concentration
functional group. ▪ From left to right, substrate increases in
o E.g., Phosphate esterase concentration

▪ No matter what molecule, for as long as it o Y axis

contains a phosphate ester, it will act on that ▪ Velocity or speed of the reaction
molecule. ▪ As you go higher, the speed increases

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 7
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

Michaelis-Menten Equation • The formula of Michaelis-Menten was reciprocated to

form the Lineweaver-Burk equation:
𝟏 𝐊𝒎 𝟏 𝟏
= +
𝑽 𝑽𝒎𝒂𝒙 [𝑺] 𝑽𝒎𝒂𝒙

o Where:
1
▪ Y axis =
𝑉
1
▪ X axis =
[𝑆]
▪ Vmax is represented by the y-intercept, where
the line of enzymatic reaction crosses the y axis
▪ Km corresponds to the x-intercept

Enzyme Concentration vs Rate of Reaction

• The graph above describes the kinetics of the enzymes

as the substrate increases. • This graph shows that
the more enzymes
o Y axis – Reaction velocity or speed added, the more active
o X axis – Substrate concentration sites → the faster the
reaction
• Michaelis-Menten equation:
o More enzyme
𝑽𝒎𝒂𝒙 [𝑺] molecules can react
𝑽=
𝑲𝒎 + [𝑺] with more substrate
molecules, so the
o This equation will obtain the Michaelis-Menten reaction rate
curve. increases
• Characteristics of the curve:
o The velocity of the reaction increases in proportion • Assumes that there is a large excess of substrate
to the concentration of the substrate. o The rate of reaction will increase only if there is
o However, when there is an excessive substrate increased substrate concentration
concentration, the velocity of the reaction can no o Less substrate and more enzymes = Rate of
longer increase → reach a maximum velocity. reaction remains the same
▪ Hence, the speed of the reaction plateaus. • This graph is used as a principle in the laboratory in
• Km (Michaelis-Menten constant) assays involving enzymes because we want to know
the concentration of enzymes.
o The substrate concentration in which its velocity is
half of the Vmax (maximum velocity)
TWO THEORIES OF ENZYME-SUBSTRATE
▪ Example: amylase, amylose, lipase and
COMPLEX
triglycerides have its own Km.

Lineweaver-Burk Plot

• A mathematical transformation of the Michaelis-Menten

curve

o Problem of
Michaelis-Menten
curve:
▪ The curve is
difficult to
read, and
difficult to
discover the
Km of a given
enzyme

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 8
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

Lock and Key Theory (1) Substrate Concentration

First-order Reaction

• Are those which proceed at a rate exactly proportional

to the concentration of only one reactant (substrate).
o Are enzymatic reactions in which the speed of the
reaction is dependent on the concentration of the
substrate; one substrate alone.
• 𝑨→𝑷
o Rate of reaction is exactly proportional to the rate of
disappearance of Substrate (A) or the appearance
of Product (P)
• Proposed by Emil Fischer
• Enzyme serves as the key and the substrate serves as Second-order Reaction
the lock
• Problem: It assumes that enzymes are rigid when in • Are those in which the rate is proportional to the product
reality, it is flexible. of the concentration of two substrates
• 𝑨 + 𝑩 → 𝑷
Induced Fit theory
o Rate of reaction is exactly proportional to the rate of
disappearance of Substrate (A) or the appearance
of Product (P)

Zero-order Reaction

• The reactions are zero-order with respect to the

reactants (substrates)
• Rate of reaction depend not on the concentration of the
substrate, but on the concentration of the enzyme.
o Rate of reaction depends on the concentration of the
• Proposed by Robert Copeland molecular species undergoing reaction
• There is a conformational change in the active site until Michaelis-Menten Curve
the substrate is complementarily fit into the active site.
o Active site if flexible.
• Lysozyme
o An enzyme that helps kill bacteria by binding to the
polysaccharide coating of the bacteria.
• Induced fit
o Change of shape of the active site
▪ The shape of the active site changes to fit the
polysaccharide substrate
▪ By initiating the “induced fit”, the enzyme breaks
• First-order reaction
the polysaccharide, which helps in killing the
bacteria. o The speed of the reaction increases as the
concentration of the substrate increases.
o Depends on the concentration of the substrate.
FACTORS THAT INFLUENCE ENZYMATIC • Zero-order reaction:
REACTIONS
o The rate of the reaction in the plateau of the graph
• Factors that affect proteins are the same factors that does not depend on the substrate concentration.
affect enzymes
• No matter how much substrate is added, the reaction
o But there are factors that are unique to enzyme will stay the same.
alone (e.g., Substrate concentration)
o The only way to increase the maximum velocity is by
• In every substrate reaction: adding more enzyme.
o Enzyme + Substrate → Enzyme-Substrate complex o Depends on the enzyme concentration
→ Enzyme + Product NOTE: To achieve the zero-order reaction in the laboratory
to measure the enzyme concentration of a patient
sample, we must increase in excess the substrate.

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 9
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

(2) Enzyme Concentration

• The higher the enzyme level,

the faster the reaction will
proceed
• More enzyme molecules can
react with more substrate
molecules, 50 the reaction
rate increases
• This scenario assumes that
there is a large excess of
substrate.
(3) pH

• pH = 7.0 - 8.0
• Often a product of the enzyme-catalyzed reaction.
o Depends on the enzyme
o When a lot of products are made, the product would
• Changes in pH may denature the enzyme compete with substrate for the enzyme’s active site,
• Protein in nature thus, slowing the reaction.
o pH affects proteins because pH (amount of • The competitive inhibitor has roughly the same shape
hydrogen ions in a solution) affects how carboxylic as the substrate, so it can fit in the active site.
acid group and the amino group in amino acids o When it is there the substrate is unable to bind to
behaves the enzyme and the desired reaction does not occur.
▪ Which is to either gain or lose hydrogen.
• A competitive inhibitor may be the end product of the
(4) Temperature reaction (negative feedback), or it may be another
chemical which blocks the substrate from binding to the
• 37°C (normal) active site.
• Denaturation at 40-50°C • Adding more substrate can decrease the inhibition
• Assay temperatures:
o More chances that the substrates will go to the
o At 25°, 30°, or 37°C active site
(5) Cofactors Noncompetitive Inhibition of Enzymes (Allosteric
Inhibition)
• Non-protein entities that must bind to particular
enzymes before a reaction occurs • Noncompetitive inhibitor inhibits the allosteric site.
• Activators: metallic or nonmetallic
o Once the connect to the allosteric site of the
o Calcium, ferrous, magnesium, manganese, zinc, enzyme, they might change the form of the enzyme
potassium, bromide, chloride ions to the point that the substrate can no longer fit to the
active site.
• If they are absent and they are needed in the enzymatic
reactions then enzymatic reactions cannot proceed
Activators

• Proper substrate binding

• Linking substrate to the enzyme or coenzyme
• Undergoing oxidation or reduction
Coenzymes

• Second substrates of enzymatic reactions

• Prosthetic group
• In assays, this should be provided in excess

(6) Inhibitors

• Interfere with enzyme reactions

o a certain molecule that would inhibit a reaction

Competitive Inhibition of Enzymes

• Competitive inhibitor competes with the substrate for

the active site.

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 10
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

• The regulatory site is often called an allosteric site. Maximum Velocity and Michaelis-Menten Constant
• Inhibiting molecule is often the product of the enzyme-
catalyzed reaction Competitive Inhibition
o Inhibition is said to be a form of a negative feedback
because an increase in the product, forces a • Black curve – uninhibited
decrease in the enzyme reaction reaction

• When in high concentrations, the allosteric inhibitor o Vmax = 1 (unchanged)

binds to the regulatory site. o Km = 1 (unchanged)

o This changes the shape of the enzyme, thus • Purple curve –

changing the shape of the active site, and thus competitively inhibited reaction
preventing the enzyme from catalyzing its reaction. o Vmax = 1 (unchanged)
o When the concentration of inhibitor decreases, the o Km = 4.0 (increased)
inhibitor leaves the regulatory site, and the active
site then resumes its normal shape. ▪ Takes more substrate to reach the maximum
velocity
▪ The enzyme resumes catalyzing its reaction
Noncompetitive Inhibition
• Adding more substrate will not decrease the
inhibition • Black curve – uninhibited
o It will only compete with the allosteric site reaction
Types of Noncompetitive Inhibitor o Vmax = 1 (unchanged)
o Km = 1 (unchanged)
• Reversible • Purple curve – noncompetitively inhibited reaction
o Once inhibitor is removed to the allosteric site, the o Vmax = 0.5 (decreased)
active site returns to normal o Km = 1.0 (unchanged)
• Irreversible ▪ Maximum velocity will not be reached if inhibited
o Once it joins to the allosteric site, it will destroy the noncompetitively.
enzyme. Uncompetitive Inhibition
Uncompetitive Inhibition of Enzymes
• Black curve – uninhibited
reaction
o Vmax = 1 (unchanged)
o Km = 1 (unchanged)
• Red curve – uncompetitively inhibited reaction
o Vmax = 0.5 (decreased)
o Km = 0.5 (decreased)
• Problem:
o Changes between competitive, noncompetitive, and
uncompetitive inhibition in the Michaelis-Menten
Curves are not apparent.
• Uncompetitive inhibitor will not inhibit unless enzyme
substrate complex is formed. • Solution: Use Lineweaver-Burk transformation line in
o It will not bind to active site nor the allosteric site of analysing enzymes to better visualize inhibitions.
the enzyme, unless there is an enzyme substrate
Lineweaver-Burk Line Interpretation
complex.
• The more substrates added, the more enzyme- • Vmax – point where the line intersects the y-axis
substrate complex will form, the more the reaction is • Km – point where the line intersects the x-axis
inhibited. • Changes in the Lineweaver-Burk Line indicates what
• Adding more substrate will result to increase type of inhibition inhibits the reaction
inhibition Competitive Inhibition
o More enzyme-substrate complex, more inhibition
• Black line – uninhibited
Summary reaction

• The more substrates added: o Vmax = 1 (unchanged)

o Km = 1 (unchanged)
o Competitive inhibition: Decreased inhibition
o Noncompetitive inhibition: Inhibition stays the same • Red line – competitively
o Uncompetitive inhibition: Increased inhibition inhibited reaction

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 11
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

o Vmax = 1 (unchanged) o If the serum of the patient is added to the reagent,

o Km = 2.5 (increased) lactate becomes pyruvate and NAD+ becomes
NADH.
▪ Slope increases but Y-intercept is still the same
▪ NAD+, overtime, increases in
Noncompetitive Inhibition
absorbance/concentration.

• Black line – uninhibited reaction o The rate of the increase in concentration

corresponds to the enzyme concentration.
o Vmax = 1 (unchanged)
o Km = 1 (unchanged) • Zero-order reaction is important because lactate and
NAD+ may run out, resulting in a plateau of the rate of
• Red line – noncompetitively the reaction
inhibited reaction
o Hence, enzyme concentration cannot be measured
o Vmax = 0.5 (decreased)
o Km = 1 (unchanged) • Time over absorbance graph
▪ Vmax (y-intercept) moves up while the Km (x- o Linear portion of the graph is where the enzyme
intercept) remains the same. concentration can be measured.
o NOTE: Lineweaver-Burk line shows a reverse ▪ If there will be too much enzyme, lactate and
interpretation of the Vmax. The higher the Vmax (y- NAD+ will be immediately utilized. The graph will
intercept), the slower the reaction velocity. show a sudden increase and becomes plateau.
▪ The enzyme concentration cannot be measured
Uncompetitive Inhibition if the substrate is inadequate. Hence, excess
substrate concentration is needed.
• Black line – uninhibited reaction
Enzymatic Assays
o Vmax = 1 (unchanged)
o Km = 1 (unchanged) Coupled-Enzyme Assay
• Red line – Uncompetitively • Substances other than substrate or coenzyme are
inhibited reaction necessary and must be present in excess.
o Vmax = 0.5 (decreased) • NAD+ or NADH
o Km = 0.5 (decreased)
o Convenient when neither is a coenzyme
▪ Shows parallel lines
• Use of auxiliary enzyme and indicator enzyme

MEASUREMENT AND ASSAYS

Measurement of Enzymatic Activity

• Ways to measure the enzyme activity in the lab:

o Increase in product concentration
o Decrease in substrate concentrate
o Decrease or increase in coenzyme concentration
o An increase in the concentration of the altered
coenzyme
[𝑬𝒏𝒛𝒚𝒎𝒆]
[𝑺𝒖𝒃𝒔𝒕𝒓𝒂𝒕𝒆] → [𝑷𝒓𝒐𝒅𝒖𝒄𝒕] • Example: Lipase Test
o In measuring the lipase of the patient, we do that in
• Enzymes can be measured by measuring either its
a series of enzymatic reactions.
substrate or product
▪ Triglycerides and Glycerokinase are part of the
o NOTE: Coenzymes can be considered as
reagent.
substrates while altered coenzymes can be
considered as products o This assay is performed in sequence because there
is no spectrophotometric analysis that could directly
• How to measure the LDH of a patient? measure glycerol and fatty acids.
𝐿𝑎𝑐𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷+ ↔ 𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 + 𝐻 +
Fixed-Time Method (End-point)
o Reagent must contain the substrates lactate and
NAD+, and must be provided in excess to achieve • Reactants are combined.
the zero-order reaction. • Reaction proceeds for a designated time.
o LDH is provided by the serum of the patient, and it is • Reaction is stopped.
measured using spectrophotometry. o Usually by inactivation (weak acid)
▪ NADH can absorb light at 340 nm, • Measurement is made of the amount of reaction has
▪ NAD+ cannot absorb light at 340 nm. occurred

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 12
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
 LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

Continuous-Monitoring Method (Kinetic Assay)

• Multiple measurements
• Absorbance change
o Increasing in absorbance
o Decreasing in absorbance
• Time intervals or continuously
• Linearity is verified and deviation is observable
Difference between Fixed-Time Method and Continuous-
Monitoring Method

• Fixed-time Method
o When the absorbance is measured only once.
• Continuous Method
o Several measurements over time
▪ Measure the absorbance over a series of period
of time (after 1 min, 2 mins, etc.)
• In enzymatic concentration, continuous method is much
better to see how fast the enzymatic reaction is.

Common Cause of Deviation from Linearity

• Very high concentration of enzymes

o In comparison to substrate
o Too much enzyme, substrates are consumed =
absorbance becomes plateau
▪ Enzyme concentration cannot be measured
• Too low substrate

Calculation of Enzyme Activity

International Unit (IU)

• Amount of enzyme that will catalyse the reaction of 1

micromole of substrate per minute under specified
conditions
• Enzyme activity

International Unit per Liter (IU/L)

• Enzyme concentration

Katal Unite (mole/s)

• Amount of enzyme that will catalyse the reaction f 1

mole of substrate per second under specified conditions
o 1.0 IU = 16.7 nkat

ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 13
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS