CHEM123
Ms. Rose Dyane F. Nunag, RMT, MPH
S.Y 2023- 2024: MIDTERMS
WEEK 9: ENZYMES & KREB’S CYCLE
ENZYME
● It is a compound usually a protein, that acts
as a catalyst for a biochemical reaction
● Cause cellular reaction to occur millions of
times faster
● Not consumed during the reaction but
merely help the reaction occur more rapidly
● Mostly, are globular proteins
● Medical Uses of Enzymes
- Used to diagnose and to monitor
treatment of certain diseases
- Appearance of these enzymes in the
blood often indicates that there is
tissue damage in an organ and that
cellular contents are spilling out into
the bloodstream
ENZYME STRUCTURE:
• Simple enzyme
- Composed only of protein
• Conjugated Enzyme
- Has a non – protein part in addition to a
protein part.
- Apoenzyme- Protein of the conjugated
enzyme
- Cofactor- Non – protein part of the
conjugated enzyme
- Holoenzyme- Biochemically active
conjugated enzyme produced from an
apoenzyme and a cofactor
- Combined apoenzyme and cofactor entity
- Coenzyme- Serves as a cofactor in a
conjugated enzyme
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NOMENCLATURE & CLASSIFICATION OF
ENZYMES
Named about the function of the enzyme, type
of reaction catalyzed and the substrate identity
1. Suffix most enzymes end in the suffix “ase”
- Ex. Urease, Sucrase, Lipase
- Exception: digestive enzymes
- Ex. Trypsin, chymotrypsin, pepsin
2. Type of reaction catalyzed by an enzyme is
often used as a prefix
- Ex. Oxidase- oxidation reaction
- Hydrolase- hydrolysis reaction
3. Identity of substrate and type of reaction
catalyzed
- Ex. Glucose oxidase, pyruvate
carboxylase, succinate
dehydrogenase
Substrate
- Reactant in an enzyme – catalyzed
reaction.
ENZYME CLASSIFICATION
MODELS OF ENZYME ACTION
Enzyme active site
- Small part of an enzyme's structure that is
actually involved in catalysis.
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 - A three – dimensional entity formed by
groups that come from different parts of the
protein chains
Enzyme- Substrate Complex
- The intermediate reaction species that is
formed when a substrate binds to the
active site of an enzyme.
LOCK- AND- MODEL
- Active site in the enzyme has the fixed,
rigid geometrical conformation.
- Substrate with a complementary geometry
can be accommodated.
INDUCED- FIT MODEL
- Enzyme’s active site is not rigid and static.
- There’s a constant change in shape
- Allows for changes in the shape or
geometry of the active site of an
- enzyme to accommodate a substrate.
- Result of the enzyme’s flexibility; it adapts
the incoming substrate.
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ENZYME SPECIFICITY
- Extent to which an enzyme’s activity is
restricted to a specific substrate, a specific
group of substrate, a specific type of
chemical bond, or a specific type of
chemical reaction.
- Degree of specificity is determined by the
active site.
1. Absolute Specificity
- Catalyze only one reaction
- Most restrictive of all specificities
- Catalase – enzyme with absolute
specificity
2. Group Specificity
- Act only on molecules that have a
specific functional group, such as
hydroxyl, amino or phosphate
groups.
- Carboxyl-peptidase is group
specific.
3. Linkage Specificity
- Act on the particular type of bond,
irrespective to the rest of the
molecular structure.
- Phosphatases hydrolyze phosphate
– ester bonds in all types of
phosphate esters
- Most general of the common
species
4. Stereochemical specificity
- Act on a particular isomer
FACTORS THAT AFFECT ENZYME ACTIVITY
● Enzyme Activity
- Measures the rate at which an
enzyme converts substrate to
products in a biochemical reaction
● Factors that affects enzyme activity
- Temperature
- pH
- Substrate Concentration
- Enzyme Concentration
1. TEMPERATURE
● Measure of kinetic energy of molecules.
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 ● Higher temperatures mean molecules are
moving faster and colliding more frequently
● Optimum temperature
- Temperature at which an enzyme
exhibits maximum activity
2. pH
● the charge on acidic and basic
amino acids located at the active
site depends on pH
● small pH changes can result in
enzyme denaturation and
subsequent loss of catalytic activity.
● Biochemical buffers help maintain
the optimum pH for an enzyme.
● Can also affect substrate, causing
either protonation or deprotonation
of groups on the substrate.
● Optimum pH
- pH at which an enzyme
exhibits maximum activity
- physiological pH ranges from
7.0 – 7.5
- Pepsin- Active in the
stomach, functions best at pH
2.0
- Trypsin- Operates in the
small intestines, function best
at pH 8.0
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3. SUBSTRATE CONCENTRATION
● Increased concentration of substrate will
obtain the enzyme activity.
● Turnover number
- Number of substrate molecules
transformed per minute by one
molecule of enzyme under optimum
conditions of temperature, pH and
saturation.
4. ENZYME CONCENTRATION
● Kept in a low number because enzymes
are not consumed in the reaction.
● The greater the enzyme concentration, the
greater the reaction rate.
ENZYME INHIBITION
● Enzyme Inhibitor
- Substance that slows or stops the
normal catalytic function of an
enzyme by binding to it.
1. Reversible Competitive Inhibition
2. Reversible Non competitive inhibition
3. Irreversible inhibition
TYPES OF ENZYME INHIBITION
REVERSIBLE COMPETITIVE INHIBITION
- Molecule that sufficiently resembles an
enzyme substrate in shape and charge
distribution that it can compete with the
substrate for occupancy of the enzymes
active site
- Remains in changed as it binds to the
enzyme's active site.
- Reversible process because it is
maintained but weak interactions
- Can be reduced by increasing the
concentration of the substrate.
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REVERSIBLE NON- COMPETITIVE INHIBITION
- Molecule that decreases enzyme activity
by binding to a site on an enzyme other
than the active site.
- Presence of this causes a change in the
structure of the enzyme sufficient to
prevent the catalytic groups at the active
site from properly affecting their catalyzing
action.
IRREVERSIBLE INHIBITION
- Molecule that inactivates enzymes by
forming a strong covalent bond to an amino
acid side – chain group at the enzymes
active site.
- Do not have structures similar to that of the
enzyme’s normal substrate.
REGULATION OF ENZYME ACTIVITY
1. A cell that continually produces a large
amount of enzyme for which substrate
concentration is always very low is wasting
energy. The production of the enzyme
needs to be “ turned off”.
2. A product of an enzyme- Catalyzed
reaction that is present in plentiful amounts
in a cell is a waste of energy if the enzyme
continues to catalyze the reaction that
produces the product. The enzyme needs
to be turned off.
ALLOSTERIC ENZYME
1. Have Quaternary Structure; (2 or more
protein chains).
2. Have 2 kinds of binding sites (for
substrates and for regulators).
3. Active and regulatory binding sites are
distinct from each other in both location
and shape.
4. Binding of a molecule at the regulatory site
causes changes in the overall three
dimensional structure of the enzyme,
including structural changes at the active
site.
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ENZYME WITH TWO OR MORE PROTEIN
CHAINS & 2 KINDS OF BINDING SITES
Regulators
- Substance that bind at the regulatory sites
of allosteric enzymes
1. Positive Regulator
- Increase enzyme activity
- The shape of the active site is
changed such that it can more
readily accept substrates.
2. Negative Regulator
- Decrease enzyme activity
- Changes to the active site are such
that substrate is less readily
accepted.
FEEDBACK CONTROL
- A process in which activation or inhibition
of the first reaction in a reaction sequence
is controlled by a product of reaction
sequence.
- Negative Feedback
PROTEOLYTIC ENZYMES & ZYMOGENS
● Proteolytic Enzymes
- Catalyzes the breaking of peptide
bonds that maintain the primary
structure of protein.
- Generated in an inactive form and
converted to active form when they
are needed.
● Zymogen
- Inactive precursor of a proteolytic
enzyme.
- Activation of a zymogen requires an
enzyme – controlled reaction that
moves some part of the zymogen
structure which changes the 3 –
dimensional structure of zymogen,
which affects active site
conformation.
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COVALENT MODIFICATION OF ENZYME
● Process in which enzyme activity is altered
by covalently modifying the structure of the
enzyme through attachment of a chemical
group or removal of a chemical group from
a particular amino acid within the enzyme
structure.
● Phosphorylation
- Process of addition of the phosphate
group to the enzyme by protein
kinases
● Dephosphorylation
- Removal of the phosphate group
from the enzyme by phosphatases.
OVERVIEW OF THE BIOCHEMICAL ENERGY
PRODUCTION
● Energy needed to run human body is
obtained from food
● Multi-step process that involves several
different catabolic pathways
● There are four general stages in the
biochemical energy production process:
- Stage 1: Digestion
- Stage 2: Acetyl group formation,
- Stage 3: Citric acid cycle
- Stage 4: Electron transport chain
and oxidative phosphorylation,
● Each stage also involves numerous
reactions
STAGE 1- DIGESTION
● Begins in mouth (saliva contains starch
digesting enzymes), continues in the
stomach (gastric juice), completed in small
intestine:
- Results in small molecules that can
cross intestinal membrane into the
blood
● End Products of digestion:
- Glucose and monosaccharides from
carbohydrates
- Amino acids from proteins
- Fatty acids and glycerol from fats
and oils
● The digestion products are absorbed into
the blood and transported to body’s cells
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STAGE 2- ACETYL GROUP FORMATION
● The small molecules from Stage 1 are
further oxidized.
● End products of these oxidations is acetyl
CoA
● Involves numerous reactions:
- Reactions occur both in cytosol
(glucose metabolism) as well as
mitochondria (fatty acid metabolism)
of the cells.
STAGE 3- CITRIC ACID CYCLE
● Takes place in inside the mitochondria
● First intermediate of the cycle is citric acid
therefore designated as Citric acid cycle
● In this stage acetyl group is oxidized to
produce CO2 and energy
● The carbon oxide we exhale comes
primarily from this stage
● Most energy is trapped in reduced
coenzymes NADH and FADH2
● Some energy produced in this stage is lost
in the form of heat
STAGE 4- ELECTRON TRANSPORT CHAIN &
OXIDATIVE PHOSPHORYLATION
● Takes place in mitochondria
● NADH and FADH2 are oxidized to release
H+ and electrons
● H+ are transported to the intermembrane
space in mitochondria
● Electrons are transferred to O2 and O2 is
reduced to H2O
● H+ ions reenter the mitochondrial matrix
and drive ATPsynthase reaction to produce
ATP
● ATP is the primary energy carrier in
metabolic pathways
● Citric acid cycle: A series of biochemical
reactions in which the acetyl portion of
acetyl CoA is oxidized to carbon dioxide
and the reduced coenzymes FADH2 and
NADH are produced
● Also known as tricarboxylic acid cycle
(TCA) or Krebs cycle:
- Citric acid is a tricarboxylic acid –
TCA cycle
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 - Named after Hans Krebs who
elucidated this pathway
● Two important types of reactions:
- Oxidation of NAD+ and FAD to
produce NADH and FADH2
- Decarboxylation of citric acid to
produce carbon dioxide
- The citric acid cycle also produces 2
ATP by substrate level
phosphorylation from GTP
● Summary of citric acid cycle reactions:
- Acetyl CoA + 3NAD + FAD + GDP +
Pi + 2H2O 2CO2 + CoA-SH +
3NADH + 2H+ + FADH2 + GTP
Step 1: Formation of Citrate
Step 2: Formation of Isocitrate
Step 3: Oxidation of Isocitrate and Formation of
CO2: involves oxidation– reduction as well as
decarboxylation
Step 4: Oxidation of Alpha- Ketoglutarate and
Formation of CO2
Step 5: Thioester bond cleavage in Succinyl CoA
and Phosphorylation of GDP
Step 6: Oxidation of Succinate
Step 7: Hydration of Fumarate
Step 8: Oxidation of L-Malate to Regenerate
Oxaloacetate
REGULATION OF THE CITRIC ACID CYCLE
● The rate at which the citric acid cycle
operates is controlled by ATP and NADH
levels
● When ATP supply is high, ATP inhibits
citrate synthase (Step 1 of Citric acid cycle)
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● When ATP levels are low ADP, ADP
activates citrate synthase
● Similarly, ADP and NADH control isocitrate
dehydrogenase:
- NADH acts as an inhibitor
- ADP as an activator.
● The electron transport chain (ETC)
facilitates the passage of electrons trapped
in FADH2 and NADH during citric cycle
● ETC is a series of biochemical reactions in
which intermediate carriers (protein and
non-protein) aid the transfer of electrons
and hydrogen ions from NADH and FADH2
● The ultimately receiver of electrons is
molecular oxygen
● The electron transport (respiratory chain)
gets its name from the fact electrons are
transported to oxygen absorbed via
respiration
● The overall ETC reaction: 2 H+ + 2e- + 1/2
O2 H2O + energy
● Energy is used to synthesize ATP in
oxidative phosphorylation
● Note that 2 hydrogen ions, 2 electrons, and
one half-oxygen molecule react to form the
product water
● This relatively straight forward reaction that
requires eight or more steps
● The reaction releases energy (exothermic
reaction)
● The energy released is coupled with the
formation of three ATP molecules per every
molecule of NADH processed through ETC
● The enzymes and electron carriers needed
for the ETC are located along inner
mitochondrial membrane
● They are organized into four distinct protein
complexes and two mobile carriers
● The four protein complexes tightly bound to
membrane:
● Complex 1: NADH-coenzyme Q reductase
● Complex II: Succinate-coenzyme Q
reductase
● Complex III: Coenzyme Q - cytochrome C
reductase
● Complex IV: Cytochrome C oxidase
● Two mobile electron carriers are:
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 - Coenzyme Q and cytochrome c.
COMPLEX 1: NADH- COENZYME Q
REDUCTASE
- NADH from citric acid cycle is the source of
electrons for this complex
- It contains >40 subunits including flavin
mononucleotide (FMN) and several
iron-sulfur protein clusters (FeSP)
- Net result: Facilitates transfer of electrons
from NADH to coenzyme Q
- Several intermediate reactions are involved
in this electron transfer
- In the final stage of electron transfer, the
electrons from cyt a3, and hydrogen ion
(H+) combine with oxygen (O2) to form
water
- O2 + 4H+ + 4e- 2 H2O
- It is estimated that 95 % of the oxygen
used by cells serves as the final electron
acceptor for the ETC

COMPLEX II: SUCCINATE- COENZYME Q

REDUCTASE
- Smaller than complex I
- Contains only four subunits including two
iron-sulfur protein clusters (FeSP)
- Succinate is converted to fumarate by this
complex
- In the process it generates FADH2
- CoQ is the final recipient of the electrons
from FADH2

COMPLEX III: COENZYME Q- CYTOCHROME-

c REDUCTASE
- Complex III contains 11 different subunits
- Several iron-sulfur proteins and
cytochromes are electron carriers in this
complex
- Cytochrome is a heme iron protein in which
reversible oxidation of an iron atom occurs
- Various cytochromes, e.g., cyt a, cyt b, cyt
c, differ from each other by:
- Their protein constituents
- The manner in which the heme is
bonded to the protein
- Attachments to the heme ring

COMPLEX IV: CYTOCHROME c OXIDASE

- Contains 13 subunits including two
cytochromes P.S. Watch the recorded discussion about enzymes & kreb’s
cycle for better understanding.
- The electrons flow from cyt c to cyt a to cyt
a3 Do not share without ACDN’s permission.

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