BIOCHEMISTRY

Enzymes
Dr. Floro B. Madarcos || 08/17/2017

OUTLINE
I. Definition of Terms VII. Enzyme Kinetics ● Elucidate the different ways of regulating enzyme activity.
A. Enzymes A. Michaelis-Menten ● Explain the factors affecting enzyme activity.
B. Apoenzyme Saturation Curve ● Elucidate uses and clinical application of enzymes.
C. Cofactor B. SIGNIFICANCE OF
D. Prosthetic group KM I. Definition of Terms
E. Holoenzyme C. Lineweaver-Burke
F. Metalloenzyme Double Reciprocal ● Enzymes - specialized protein catalysts that accelerate
G. Regulatory enzyme Plot chemical reactions in biological system to about 105 to
H. Active site VIII. Enzyme Inhibition 1014 times faster than uncatalyzed reactions; not
I. Allosteric site C. Reversible consumed after the reaction.
J. Substrate D. Irreversible  Enzyme catalysts bind reactants (substrates),
II. Enzyme Cofactors IX. Enzyme Activity convert them to products, and release the products.
A. CoA Regulation Although enzymes may be modified during their
B. NAD+ A. Feedback Inhibition participation in this reaction sequence, they return to
C. Biotin B. Allosteric their original form at the end.
D. TPP Modification  Almost every biochemical reaction is catalyzed by
E. FAD C. Covalent Modification an enzyme.
F. PLP D. Zymogen  With the exception of a few catalytic RNAs
G. Inorganic Coenzymes Modification (ribozymes), all known enzymes are proteins. Many
III. Six Major Classes of E. Induction or require non-protein coenzymes or cofactors for their
Enzymes Repression catalytic function
A. Oxidoreductases F. Synthesis and ● Apoenzyme – or apoprotein, a protein component of
B. Transferases Degradation enzyme, without cofactors or prosthetic groups;
C. Hydrolases G. Protein-Protein catalytically inactive
D. Lyases Interaction ● Cofactors - small, soluble non-protein component
E. Isomerases X. Factors Affecting required for apoenzyme activity; reversibly bound to
F. Ligases Enzyme Activity enzyme and participates in overall catalytic activity; helps
IV. Characteristic of A. Temperature bind substrate and catalyze its chemical transformation
Enzymes B. pH into product
V. Models of Enzyme- C. Substrate  Coenzyme – small organic molecules often derived
Substrate Complex Concentration from vitamins; bound covalently or loosely to the
A. Lock and Key Model XI. Enzymes in Clinical apoenzyme
B. Induced Fit Model Diagnosis  Prosthetic Group – organic molecule firmly or
VI. Enzyme-Catalyzed XII. References permanently attached to apoenzyme
Reactions XIII. Quiz  Metal Ions – inorganic molecules (K+, Fe2+, Mg2+)
● Holoenzyme - enzyme with its nonprotein component;
OBJECTIVES apoenzyme + cofactor; catalytically active
● Metalloenzyme – enzymes that require a metal in their
At the end of the lecture the students must be able to: composition; bind to and retain metal atoms under all
● Define the following:
conditions due to high affinity (2020C)
 Enzymes
● Regulatory Enzyme – catalyzes rate limiting or
 Apoenzyme committed step or the slowest step of a metabolic
 Cofactor pathway; usually irreversible; regulates the pathway
 Prosthetic group activity (Table 1)
 Holoenzyme  Activity is affected by several factors (positive and
 Metalloenzyme negative) so that an increase or decrease in
 Regulatory enzyme enzyme's activity rate changes rate of entire
 Active site metabolic pathway
 Allosteric site ● Active site of an enzyme – site where substrate binds;
 Substrate where the enzyme–substrate complex (ES complex)
● Discuss the helpers (cofactors) of enzymes. forms to catalyze a chemical reaction; located in clefts or
● Enumerate the six major classes of enzymes. crevices
● Discuss the characteristics of enzymes.  Contains functional groups (donated by the
● Compare the two models of enzyme-substrate complex. polypeptide chain, or by bound cofactors) that
● Discuss the operation and plots used to illustrate enzyme directly participate in the reaction.
kinetics. ● Allosteric site of an enzyme - are sites other than the
● Differentiate ways of enzyme inhibition and their effects on active site; Allosteric activators or inhibitors regulate the
reaction velocity. enzyme through conformational changes affecting the
 Reversible catalytic site causing this to be more active or less active
 Irreversible ● Substrate - reactant; molecule acted upon by the enzyme
to form a product

Trans #4 Group #19 : Cenina, Chan A., Chan S., Escobia, Espeleta Trans Head: Caballar 1 of 10
 Thiamine Pyruvate Aldehydes Thiamine
Table 1. Summary of regulatory enzymes. Pyrophosphate dehydrogenase (Vit. B1)
(TPP) Isocitrate
Enzymes Process Positive Negative Catalyzes dehydrogenase α-
ketoglutarate
Phospho Glycolysis AMP, F- ATP, Phosphorylatio dehydrogenase
fructokinase 2,6-bisPO4 Citrate, H n of fructose 6- Transketolase, α-
1 phosphate Ketoacid
fructose 1, 6-
dehydrogenase
bisphosphate
Flavin Adenine Succinate Electrons Riboflavin
Dinucleotide dehydrogenase α- (Vit. B2)
Acetyl De Novo Citrate, Malonyl Carboxylation (FAD) Ketoglutarate
Cholinestera Synthesis Insulin, CoA, acetyl CoA into dehydrogenase
se of Fatty High CHO, Palmityl malonyl CoA Pyruvate
dehydrogenase
Acids Low Fat, CoA,
Nitric oxide
High Epinephri
synthase
Protein ne,
Glucagon,
Pyridoxal Glycogen Amino groups Pyridoxine
High Fat, Phosphate phosphorylase γ- (NH3) (Vit B6)
Fasting (PLP) ALA synthase,
Histidine
HMG COA Cholester Insulin, T3, Cholester Reduction of decarboxylase
Reductase ol Glucocortic ol, Bile HMG CoA into Alanine
Synthesis oid Acids, mevalonate aminotransferase
Mevalonat
e Lipoate Pyruvate Electrons and Not required
dehydrogenase α- acyl groups in diet
Glucose 6 P Pentose Conversion of Ketoglutarate
Dehydrogen Phosphat glucose 6-
dehydrogenase
ase e phosphate into
5’deoxycobalamin Methylmalonyl H atoms and Cobalamin
Pathway 6phosphogluc
mutase alkyl groups (Vit B12)
onolactone
Tetrahydrofolalate Thymidylate One-carbon Folic acid
II. Enzyme Cofactors (FH4; TH4) synthase groups (Vit.B9)

● They are complex nonprotein organic molecules that COENZYME A (COA OR COASH)
participate in catalysis by providing functional ● Pantothenic acid – derived; cofactor of several enzymes
groups,much like the amino acid side chains like acetyl CoA carboxylase
● They are either coenzymes (usually vitamin derivatives), ● Takes part in reactions of the TCA, FA synthesis and
prosthetic groups, or metal ions (metalloenzymes). oxidation, acylations and cholesterol synthesis
● They are temporary carriers of specific functional groups ● Examples:
during metabolism. α – Ketoglutarate Dehydrogenase Complex
● They are molecules attached to the apoenzyme to make
it catalytically active. (cofactor + apoenzyme =  α – Ketoglutarate → Succinyl CoA
holoenzyme  Enzymes: Pyruvate decarboxylase, dihydrolipoyl
● Very little activity and specificity in absence of enzyme transacetylase, dihydrolipoyl dehydrogenase
 Succinyl CoA receives strong thioester bond from
Table 2. Summary of coenzymes. CoA NAD+ reduced to NADH Irreversible and redox
COENZYME ENZYME CHEMICAL VITAMIN reaction of TCA
ATTACHED TO GROUPS PRECURSOR Pyruvate Dehydrogenase Complex
TRANSFERRED
 Pyruvate → Acetyl CoA
 Enzymes: Pyruvate decarboxylase, dihydrolipoyl
Coenzyme A Acetyl CoA Acyl groups Pantothenic
transacetylase, dihydrolipoyl dehydrogenase
(CoASH) carboxylase acid (B5) &
other  Other factors involved: TPP, Lipoate, FAD Part of
compounds Glycolysis

Nicotinamide Lactate Hydride ion Nicotinic acid NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD+)
Adenine dehydrogenase (:H-) (Niacin; B3) ● Coenzymes of dehydrogenases involved in REDOX
Dinucleotide Other reactions
(NAD) dehydrogenase ● NAD+ reduced to NADH + H+
● Example:
Biocytin Pyruvate CO2 Biotin (B7)  Lactate → Pyruvate
carboxylase,
 Transfer of 2H+ ions from lactate to NAD+
Acetyl CoA
carboxylase,  Enzyme: Lactate Dehydrogenase
Propionyl CoA
carboxylase

BIOCHEMISTRY ENZYMES 2 of 10
 ● Zinc
BIOTIN  CO2 → Carbonic acid (H2CO3) through hydration
● Important in carboxylation reactions
● Carrier of activated CO2 in form of bicarbonate (H2CO3)  Enzyme: Carbonic anhydrase
● One of the B – complex vitamins (B7) Table 3. Summary of Inorganic Cofactors
● Covalently attached to a lysine residue on the nitrogen
portion of the carboxylase enzyme INORGANIC COFACTORS ENZYMES
● Example:
 Pyruvate → Oxaloacetate Zn+2 Carbonic anhydrase, Alcohol
dehydrogenase,
 Enzyme: Pyruvate Carboxylase Carboxypeptidases A & B
 Acetyl CoA → Malonyl CoA
 Enzyme: Acetyl CoA carboxylase (Biotin) (De Novo Cu+2 Cytochrome oxidase
Synthesis of Fatty Acids)
Mn+2 Arginase, Ribonucleotide
THIAMINE PYROPHOSPHATE (TPP)
reductase
● Vitamin B1 derived cofactor in energy yielding
metabolism
Mg+2 Hexokinase, Pyruvate kinase,
● Functional group is the reactive carbon o Carrier of
Glucose 6-phosphatase
aldehyde group
● Example:
Ni+2 Urease
 Pyruvate → Acetyl CoA
 Enzyme: Pyruvate Dehydrogenase Mo Nitrate reductase
● Plays a special role in:
 Transketolase Reactions in Pentose Phosphate Se Glutathione peroxidase
Pathway
 Oxidative decarboxylations in Citric Acid Cycle K+ Propionyl CoA carboxylase
 Catabolism of the branched-chain amino acids

FLAVIN ADENINE DINUCLEOTIDE (FAD) III. Six Major Classes of Enzymes

● Riboflavin (Vit. B3) derived cofactor of several
dehydrogenases involved in REDOX reactions O-xidoreductases L-yases
● FAD+ reduced to FADH2
● Example:
T-ransferases I-somerases
 Succinate → Fumarate
 Enzyme: Succinate Dehydrogenase H-ydrolases L-igases
● Plays a special role in:
 Citric Acid Cycle
 Nitric Oxide Synthesis (with Flavin mononucleotide- Mnemonic: OTHLIL
FMN)
 ß-oxidation of fatty acids Table 4. Summary of Enzyme Classes
CLASS SUBCLASS ENZYME EXAMPLES
PYRIDOXAL PHOSPHATE (PLP) )
● Major enzyme in transamination reactions
Oxidases Dehydrogenases Transfer of Lactate
● Vit. B6 derived cofactor involved in carbohydrate, amino acid
, Oxidases, electrons dehydrogenase
and neurotransmitter synthesis
● PLP transfers AAs via functional group of reactive aldehydes Reductases, and L-lactate <-> Pyruvate
Peroxidases, hydrogen
 Forms covalent intermediate with amino groups of AAs
Catalases, atoms from
● Example:
Oxygenases, donors to
 Glycine + Succinyl CoA → ALA Hydroxylases acceptors
 Enzyme: δ – Aminolevulenate synthase LEO – Loss
● Plays a special role in: of electrons
 Carbohydrate metabolism - Glycogenolysis (oxidation)
 Amino Acid Metabolism - Heme Synthesis, Histamine GER – Gain
Synthesis,Transamination, Neurotransmitter Synthesis of electrons
(reduction)
METAL IONS OR INORGANIC MOLECULES (Iron-sulfur clusters)
● Magnesium Transferases Transaldolase Transfer Alanine transaminase
and functional Kinase – transfers
 Reaction: Glucose → Glucose – 6 – phosphate
Transketolase: groups from phosphate from ATP to
 Enzymes: Glucokinase, Hexokinase acyl, methyl, donors to an acceptor
 Mg2+ bound to 3 phosphate groups of ATP making it glucosyl, and acceptors; -Hexokinase and
easy for kinases to attach to and transfer them to phosphoryltransf utilize 2 Glucokinase in
G6P erases, Kinases, substrates hydrolysis
 Kinases usually require Mg2+ as a cofactor Phosphomutase to produce 2 Glycosyltransferase –
● Potassium s, products if the transferred group
Transaminases is a carbohydrate
 Phosphoenolpyruvate (PEP) → Pyruvate residue
 Enzyme: Pyruvate kinase -Galactosyl

BIOCHEMISTRY ENZYMES 3 of 10
 C. They increase reaction rates by lowering activation
transferase in
energy.
Sphingolipid Synthesis
a. Activation energy: energy needed to form a
transition state; difference in free energy
Hydrolases Esterases, Catalyze Pyrophosphatase
Glycosidases, cleavage of Thiolase in ß-oxidation between the transition state and substrate
Peptidases, chemical of Fatty Acids b. Transition state: complex of reactants
Phosphatases, bonds by competent to produce reaction products. It
Thiolases, additional of has a higher free energy than substrate and
Phospholipases, H2O product.
Amidases, producing 2 c. Enzyme binds to the transition-state
Deaminases, products structure thereby decreasing the activation
Ribonucleases
energy.
Lyases Decarboxylases, Cleave C-C, Pyruvate
Aldolases, C-O, C-N decarboxylase
Hydratases, bonds by Cystathionine
Dehydratases, means other synthase
Synthases, than Neurotransmitter
Lyases hydrolysis Synthesis:
or oxidation -DOPA
Catalyze C- decarboxylase
C bond -Histidine
cleavage in decarboxylase
reversible Glycolysis:
reaction -Aldolase A
Krebs Cycle:
-Citrate Synthase,
Fumarase
(or fumarate hydratase)
Synthase – catalyze
reactions that favors
formation of a C-C
bond
Hydralases – add H2O
to a substrate

Isomerases Epimerases, Transfer of Alanine racemase

Isomerases, functional Glycolysis:
Mutases, groups or -Triosephosphate
Racemases double bond isomerase
within same -Phosphohexo
molecule isomerase
-Phosphoglycerate
mutase
Pentose Phosphate
Pathway: -
Epimerase

Ligases Synthetases, Catalyze Gluconeogenesis:

Carboxylases joining of -Pyruvate
substrates carboxylase
in presence Urea Cycle: Figure 1. Transition states -- catalyzed vs. uncatalyzed. Changes in energy from
of ATP -Arginosuccinate Substrate to Product (Above) and Involving Intermediate products (Below)
synthetase
Heme Synthesis: D. They are highly specific for the reactants or
-δ-ALA synthase substrates they act on.
a. Substrates are bound to specific substrate-
binding sites on the enzyme through
IV. Characteristic of Enzymes interactions with the amino acid residues of the
enzyme. This ensures that each enzyme is
A. Enzymes are not changed by the reaction they selective for its substrates. Hence, only specific
catalyze. products are formed.
a. Although, they may be temporarily be changed E. Enzymes are mostly protein in nature.
in the middle of the reaction. a. Enzyme are made up of chains of amino acids
b. They don’t make new reactions, they just speed joined together by a peptide bond.
them up. b. Their catalytic activity depends on the integrity
B. Enzymes don’t change the equilibrium position of the of their native protein conformation.
reaction. c. The protein’s shape may change when
a. Enzymes can’t force non-energetically- denatured, becoming dysfunctional. The
favorable/ non-spontaneous reactions. primary structure does not change in denature.
b. The time it takes to achieve equilibrium is
significantly less with an enzyme.

BIOCHEMISTRY ENZYMES 4 of 10
 V. Models of Enzyme-Substrate Complex complex in a relatively fast reversible step.
(Lehninger’s)
A. Lock and Key Model
● Fisher’s Template Theory (Emil Fischer, 1894)
● Binding site on the enzyme complements the
substrate
● Does not take into account the three-dimensional
flexibility of proteins

where:
E - Enzyme
S - Substrate
ES - Enzyme-substrate complex/ Michaelis complex
K1 - Rate constant

● Second Step
 ES complex then breaks down in a slower
second step to yield the free enzyme and the
reaction product
Figure 2. Schematic diagram of Fischer’s lock and key model  The overall rate must be proportional to the
concentration of the species that reacts with the
B. Induced Fit Model ES because the slower second reaction must
● Koshland’s Induced Fit Model (Daniel E. limit the rate of the overall reaction. (Lehninger’s)
Kosherland, Jr., 1958)
● Enzyme undergoes a slight conformational change
when it binds to the substrate.(substrate induces the
change in conformation)
● Through hydrogen bonds, the enzyme’s active site
molds around the substrate to compliment it.

where:
P - Product
K2 - Rate constant

● Overall Reaction

Figure 3. Schematic diagram of Koshland’s induced fit model

VI. Enzyme Catalyzed Reactions

VII. Enzyme Kinetics
A. KINETICS
● Study of reaction rates (velocity)
A. MICHAELIS – MENTEN EQUATION
● Kinetic measurements on enzyme-substrate
● It is the rate equation for one substrate, enzyme
reaction systems are used to study the mechanism
catalyzed reaction
of enzyme-catalyzed reactions. These include
● Illustrates in mathematical terms the relationship
studying the rates of reaction at different substrate
between initial velocity and substrate concentration
and enzyme concentration.
● Useful in measuring:
 concentration of enzyme in a mixture using its
catalytic activity
 Purity
 Catalytic efficiency and/ or specificity for different
substrate Vo = Initial Velocity
 Comparison of isomers Vmax = Maximal velocity
 Effects of inhibitors Km = Michaelis constant (concentration of substrate required
B. EFFECT OF SUBSTRATE CONCENTRATION IN to reach 1/2 Vmax
ENZYME KINETICS [S] = Substrate concentration
● First Step
 Michaelis and Menten postulated that the
enzyme first combines reversibly with its
substrate to form an enzyme-substrate

BIOCHEMISTRY ENZYMES 5 of 10
 has a KM value of 5 mM. Thus, Glucokinase has
less affinity for glucose compared to Hexokinase
● When blood glucose is low (ex: occurs in fasting
state or during starvation), hexokinase is used to
phosphorylate glucose into glucose-6-phosphate.
The brain is therefore assured of its energy needs
even in times of glucose depletion.
● Glucokinase, on the other hand is only active when
blood glucose is high (ex: immediately after a meal
because glucokinase affinity for glucose is high only
if there is elevated blood glucose)
● The high KM of hepatic glucokinase thus promotes
the storage of glucose as liver glycogen or as fat in
Figure 4. Michaelis-Menten Saturation Curve. adipose tissues (only when there is an excess in
glucose supply).
● Graphical representation of the Michaelis-Menten
Equation C. LINEWEAVER – BURKE RECIPROCAL PLOT
● A hyperbolic curve is observed when Vmax is
reached asymptomatically
● Reaction velocity (V0) linearly increases as [S]
increases and begins to level off and approaches a
maximum at higher substrate concentrations (Vmax)
● At Point A, [S] < Km. Only a portion of the total
number of enzyme active sites are saturated by the ● Linearizes the Michaelis-Menten Equation and is a
substrate and can be found as ES more precise way to measure Vmax and Km of an
● At Point B, [S] = Vmax/2. Half of the total number enzyme; Useful in analysis of enzyme inhibition and
of enzyme active sites are saturated and are at the distinguishing between various enzymatic reactions
ES complex at any instant. mechanisms
● At Point C [S] > Km. Velocity increases with
substrate concentration until it reaches a plateau
where velocity of reaction reaches Vmax. At this
point further increase in [S] will no longer affect the
velocity because all enzyme active sites are already
saturated with the substrate

B. SIGNIFICANCE OF KM
● Km - characteristic of an enzyme and its particular
substrate; reflects the affinity of the enzyme for that
substrate. It is the substrate concentration at which
half of the active sites of the enzyme are filled up.
● ↓Km --> ↑affinity of the enzyme for substrate --> ↓
concentration of substrate needed to achieve Vmax Figure 6. Lineweaver-Burke Reciprocacl Plot

● ↑Km --> ↓ affinity of the enzyme for substrate --> ● y axis (ordinate) - Where the reciprocal of reaction
↑concentration of substrate needed achieve Vmax. velocity 1/V, is plotted
● x axis (abscissa) - Where the reciprocal of
substrate concentration, 1/[S], is plotted.
● Km/Vmax - Slope of the line

Figure 5. Km and its Physiological Utilization of Glucose.

● Hexokinase (found in extrahepatic cells, including

RBC’s) has a KM value of 0.1 mM for glucose, Solution: Y-intercept = 1/Vmax X-intercept = - 1/Km
whereas glucokinase (found in the liver, and Vmax = 1/Y-intercept Km = - 1/x-intercept
pancreatic β-cells - an isoenzyme of hexokinase) Vmax = 1/10(mM sec-1)-1 Km = - 1/-0.3
Vmax = 0.1 mM/sec Km = 3.33 mM
BIOCHEMISTRY ENZYMES 6 of 10
 VIII. Enzyme Inhibition

A. REVERSIBLE INHIBITION
 The inhibitor binds to enzyme through non-covalent
bonds, thus dilution of the enzyme–inhibitor complex
results in dissociation of the reversibly bound inhibitor,
and recovery of enzyme activity

Figure 8. Graphical Representation of the different types of inhibition.

Types of Irreversible Inhibitors:

1. Group-specific Covalent Modifying Agents – React with
Figure 7. Representation of Reversible Inhibitions. From left to right: specific type of enzyme functional group (Ser-OH or His-
Competitive, Normal, Noncompetitive. imidazole). Eg: DIPF on ACHesterase on gap junctions
2. Affinity Labels – structurally similar to substrates ―guides
1. Competitive Inhibitor - binds to the substrate in the active site reagent to active site. Eg: Tosyl phenylalanyl
thus blocking access by the substrate; Also called substrate chloromethylketone (TPCK) on chymotrypsin
analog because they tend to resemble the structure of the 3. Transition State Analogs – structurally similar to transition
substrate; Example: lovastatin, malonate statem which binds even more tightly to enzymes than
 Kinetic Effect: Vmax unchanged; Km increased (more substrates ↑affinity to active site. Eg:
substrate needed to get to a given rate in the presence of 4. Suicide Inhibitors – enzymes treats it as a substrate, starts
inhibitor) chemical process with inhibitors. Chemical mechanism
2. Non – competitive Inhibitor - binding of the inhibitor does not itself leads to enzyme to react covalently with inhibitor =
affect the binding of the substrate; Efficiency of the enzyme to committing suicide. Eg; Penicillin on transpeptidase
transform substrate into product is decreased; Inhibitor-binding (required for bacterial cell wall synthesis)
site is physically distinct from substrate-binding site; Example:
Diisopropylphosphofluoridate (DIPF) IX. Enzyme Activity Regulation
 Kinetic effect: Vmax decreased (since the inhibitor
reduced the concentration of ES complex that can A. Feedback Inhibition
advance to reaction products); Km unchanged (since ● End product of pathway synthesis inhibits further
substrate can still bind to the enzyme production of product by inhibiting the first enzyme
3. Uncompetitive Inhibitor - Substrate binding modifies enzyme of the pathway.
structure making the inhibitor – binding site available; Rare ● Occurs since synthesis requires a lot of ATP which
occurrence would diminish the ATP reserve for other metabolic
 Kinetic effect: Vmax and Km is decreased processes
B. IRREVERSIBLE INHIBITION
 Covalent modification occurs at the active site of the
enzyme; Inhibitors chemically modify the enzyme.
Removal of the inhibitor will not reverse the effect because
the enzyme has already been poisoned

Table 5. Representative Drugs That Inhibit Specific Enzymes.

Figure 9. Schematic diagram of a feedback inhibition where the product F

inhibits enzyme 1 upon synthesis

B. Allosteric Modification
1. POSITIVE/STIMULATORY allosteric molecules
enhances enzyme activity
2. NEGATIVE/INHIBITORY allosteric molecules inhibit
enzyme activity by changing the activity site’s
conformation

BIOCHEMISTRY ENZYMES 7 of 10
 F. Control of the rates of enzyme synthesis and
degradation
● ↑ Enzyme Amount - may be related to increased
enzyme synthesis
● ↓ Enzyme Amount - may be related to decreased
enzyme synthesis
G. Protein-protein interaction
● Enzymes formed from many protein subunits may
be present in its inactive form due to its interaction
with its subunits. Activation occurs following its
separation from its regulatory subunits.
Figure 10. Schematic diagram of allosteric modification

C. Covalent Modification
X. Factors Affecting Enzyme Activity
● Phosphorylation or dephosphorylation of amino acid
1. TEMPERATURE
residues to activate or inactivate the enzyme
● the higher the temperature, the higher an
● general rule: most anabolic or biosynthetic enzymes
enzyme-catalyzed reaction’s velocity will be.
are activated when dephosphorylated and
● velocity will keep increasing until optimum
inactivated when phosphorylated
temperature (reaction velocity is at maximum) is
reached.
● 44-55°C: optimum temperature for most human
enzymes
● The higher the collision rate between enzyme
and substrate, the higher the rate of binding. The
increased temperature causes vibrational or kinetic
energy, which increases the velocity of collision that
leads to reaching the optimum temperature.
● Beyond the optimum temperature, the enzyme’s
secondary and tertiary structures start to denature,
therefore causing a decrease in overall velocity.

Figure 11. Schematic diagram of covalent modification

D. Zymogen Modification
● Zymogen - Inactive precursor of enzymes
● Cleavage of zymogen results in its activation
● example: blood coagulation
E. Induction or Repression
● INDUCTION - increase in rate of enzyme synthesis
by substances called inducers
 Constitutive enzymes - enzyme concentration
independent of inducers
 Inducible enzymes - enzyme concentration
dependent on the presence of inducers
● REPRESSION (also known as feedback regulation) -
repressors are usually end products of biosynthetic Figure 13. Effect of temperature to enzyme activity where optimum
reaction temperature is at ~44-55°C
 Repressors - low molecular weight substances
that decrease enzyme synthesis at the level of ● Plotting temperature vs. rate of reaction in a plot, a curve
gene expression SLIGHTLY SKEWED TO THE RIGHT is seen.
● To the left of the optimum temperature, the temperature
isn’t enough to provide kinetic energy to overcome
activation; to the right, the enzymes are denatured.
(Devlin)
● Clinical correlation / further reading: Thermal Lability of
Glucose-6-Phosphate Dehydrogenase Results in
Hemolytic Anemia (Devlin)

For most enzymes the optimum temperature eis about 30⁰C

A few bacteria have enzymes tha can withstand very high
temperatures even about 100⁰C
Most enzymes however are fully denatured at 70⁰C
Figure 12. Example of induction or repression of enzyme synthesis

BIOCHEMISTRY ENZYMES 8 of 10
2. PH CONCENTRATION
● The velocity of an enzyme-catalyzed reaction
increases with an increase in pH until the optimum pH
is reached.
 optimum pH - velocity of the reaction is highest,
enzyme is most active.
● Before the optimum pH, the velocity is increasing due to:
 ionization of the specific functional groups of the
amino acids that line the active or catalytic site of
the enzyme (or in the substrate)
 General formation of Hydrogen bonds important to
the overall conformation of the enzyme.
● Beyond the optimum pH, the velocity decreases due to the
denaturation of the enzyme.
 the structure of the catalytically active site of the
protein molecule depends on the ionic character of
the amino acid side chains (Madarcos)
 deprotonation of the amino (NH3+) terminal and
the alteration of the conformation of the active site of
the enzyme results to decrease in enzyme activity.

Figure 14. Effect of pH to enzyme activity where optimum pH is at 7.4

● plotting pH - enzyme velocity, the curve is BELL-SHAPED.

● Clinical correlation / further reading: Alcohol
Dehydrogenase Isoenzymes with Different pH Optima
(Devlin)

3. SUBSTRATE CONCENTRATION
● “The velocity of a reaction increases as the substrate Figure 15. Effect of substrate concentration to enzyme-catalyzed reaction
velocity. A. Ideal substrate vs reaction velocity B. Actual Relationship
concentration increases until Vmax (maximal velocity)
is reached.”
● the substrate concentration-velocity curve will give a
● Increasing substrate concentration [S] after Vmax is
reached won’t affect the reaction velocity due to the HYPERBOLIC shape. (Figure 14.a)
―saturation‖ of all the substrates in the reaction. (Hyperbolic ● Sometimes, substrates compete with each other that they
● Addition of excessive amounts of substrate, on the other forgot about the active site, thus reaction velocity tends to be
hand, would decrease reaction velocity because there slightly variable ( Figure 14.b) – Prof. Madarcos
would already be too much substrates competing for the
enzyme surfaces that they already block the activation XI. Enzyme in Clinical Diagnosis
sites. This prevents any substrate molecules from
occupying them, hence, all of the enzymes present end up CASE 1: SARIN GAS ATTACK
not being used.
In March 30, 1995, the Aum Shinrikyo terrorist group undertook
. five-coordinated release of sarin gas at the peak of the morning
Enzyme Concentration rush hour on several subway commuter train lines of Tokyo Metro.
– The increase in
enzyme concentration 13 persons were killed, fifty severely injured and thousand others
is proportional to the developed temporary vision problems.
velocity of reaction,
provided that there will 189 members of the terrorist/religious group were indicted, 13 were
be unlimited substrate, sentenced to death, 5 were given life imprisonment, 80 were given
the graph is LINEAR. prison sentences of varying lengths, 87 received suspended
sentences,12 were fined and 1 was found not guilty.

BIOCHEMISTRY ENZYMES 9 of 10
SARIN GAS EFFECTS d. Less enzyme-substrate complexes are formed.
● Essentially, sarin gas is a series of chemicals that blocks
acetylcholinesterase. It is similar to pesticides and has 5. Enzymes as biological catalysts speed up biochemical reactions
DIPM as its active component. by:
● Sarin irreversibly binds with the Ser residue in a. Changing the equilibrium constant of the reaction.
acetylcholinesterase, causing its inactivation. b. Changing the concentration of the reactants and
● Without the degradation of acetylcholine in the body, a products.
predominance of autonomic effects (uninhibited muscle c. Increasing the standard free energy change of the
contraction) will occur and will have effects leading up to reaction.
death. Specific cause of death in sarin gas victims is d. Lowering the free energy of activation of the reaction.
respiratory failure. (How Stuff Works)
answers: C;D;B;A;D
CASE 2: GOUTY ARTHRITIS

A 50-year old male, obese, and hypertensive consulted

because of extreme tenderness and swelling of the right big toe
associated with limping gait.
History revealed moderate alcohol intake of 10 years. He
is fond of eating shellfish and organ meats during drinking
sessions with friends.
His blood uric acid level was elevated. He was given
Allopurinol and was advised drastic lifestyle modification.

GOUTY ARTHRITIS
● Purine Metabolism disorder that occurs when
undegraded uric acid forms crystal deposits in joints,
tendons, and surrounding tissues.
● Allopurinol is used as treatment — inhibits xanthine
oxidase to decrease uric acid production in the body
REFERENCES
1. 2020C Biochemistry Trans
2. 2020B Biochemistry Trans
3. Dr. Madarco‟s lecture/ppt
4. Devlin -- Textbook of Biochemistry with Clinical
Correlations, 4th Ed.
5. http://science.howstuffworks.com/sarin2.htm
QUIZ
1. Which of the following is TRUE when a substrate concentration
equals Km in an enzyme-catalyzed reaction?
a. A few of the enzyme molecules are present as ES
complex.
b. Majority of the enzyme molecules are present as ES
complex.
c. Half of the enzyme molecules are present as ES
complex.
d. All of the enzyme molecules are present as ES complex.

2. Competitive inhibition can be relieved by increasing which of the

ff?
a. Enzyme concentration
b. Inhibitor concentration
c. Enzyme-substrate concentration
d. Substrate concentration

3. Which of the ff enzymes requires biotin as a coenzyme?

a. PEP carboxykinase
b. Pyruvate carboxylase
c. Phosphofructokinase I
d. Pyruvate dehydrogenase

4. An enzyme with a low Km indicates which of the ff?

a. High affinity for the substrate
b. Requires increased amount of substrate to become
saturated
c. Vmax can be reached at high substrate concentration
BIOCHEMISTRY ENZYMES 10 of
10