BIOCHEMISTRY 1.

03
JUNE 27, 2013
Enzymes
Daniel D. Menorca, M.D.

TOPIC OUTLINE 6. Substrate

 Molecule acted upon by the enzyme to form the
Outline product
I. Enzyme  For reversible reactions, products of the forward
 Definition of Terms reaction become substrate for the reverse
 Classification of Enzymes
reaction
II. Enzyme Kinetics
7. Substrate binding site
 Introduction
 Order of Reaction  Particular region of the enzyme surface where
 Michaelis-Menten Equation specificity resides
 Inhibition of Enzyme Activity 8. Active site
 Multisubstrate Reactions  Found in the substrate binding site
III. Mechanism of Enzyme Action  Contiguous to the substrate binding site in primary
IV. Clinical Applications sequence or lie in the distal regions of the primary
V. Regulation of Enzymatic Activity
sequence
VI. Review Questions
VII. Answer to Review Questions  Brought adjacent to the substrate-binding site by
folding in the tertiary structure

ENZYMES 9. Allosteric site

 Region in the enzyme that is not at the active site
DEFINITION OF TERMS or substrate binding site
 Unique site where small molecules bind and affect
1. Enzymes a change in the substrate binding site or active site
 Proteins that function in the acceleration of
chemical reactions in the biological systems
 As catalyst, they increase the rate of chemical
reaction
 Temporarily covalently bound to a molecule but at
the end will again be in their original form
 THEY DO NOT CHANGE THE EQUILIBRIUM
CONSTANT OF THE REACTION BUT SIMPLY
INCREASE THE RATE AT WHICH THE REACTION
REACHES EQUILIBRIUM
2. Rate/velocity
10. Isozyme
 Speed of chemical reaction
 Enzymes variants that catalyze the same chemical
o Rate - change in the amount (moles) of
reaction
starting materials or products per unit
time
CLASSIFICATION
o Velocity - change in the concentration of
starting materials or products per unit  The commonly used names for most enzymes describe the
time reaction catalyzed, followed by the suffix –ase.
3. Apoenzyme Examples:
 Dehydrogenases remove hydrogen atoms.
 Protein part of an enzyme without any cofactors
or prosthetic groups  Proteases hydrolyze proteins.
 Isomerases catalyze rearrangements in configuration.
 Inactive enzyme
4. Cofactors  Modifiers may precede the name to indicate:
 The substrate, e.g. xanthine oxidase
 Organic or inorganic molecules
 The source of enzyme, e.g. pancreatic ribinuclease
 Required by apoenzyme for its activity
 It regulation, e.g. hormone-sensitive lipase
 Tightly bound cofactors to an apoenzyme are
 Feature of its mechanism of action e.g. cysteine
called prosthetic group
protease
 Loosely bound cofactors are called coenzymes
 Alphanumeric designators are added to identify multiple
5. Holoenzymes
forms of an enzyme.
 Apoenzyme together with its cofactor
 Examples are RNA polymerase IIII and protein kinase
 Complete and catalytically active Cβ

International Union of Biochemists (IUB) – developed an

unambiguous system of enzyme nomenclature.
 In this system, each enzyme has a unique name and
code number that identify the type of reaction
catalyzed.
 Enzymes are grouped into six classes

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 BIOCHEMISTRY Cell and Cell Membrane: A Biochemical Approach
1. Oxidoreductases – catalyze oxidations and reductions
2. Transferases – catalyze transfer of moieties such as
glycosyl, methyl or phosphoryl groups
3. Hydrolases – catalyze hydrolytic cleavage of C-C, C-O,
C-N, and other bonds
4. Lyases – catalyze the cleavage of C-C, C-O, C-N, and
other bonds by atom elimination, leaving double
bonds.
5. Isomerases – catalyze geometric or structural changes
within a molecule
6. Ligases – catalyze the joining of two molecules
coupled to the hydrolysis of ATP
ENYZME KINETICS
The field of biochemistry concerned with quantitative
measurement of the rates of enzyme-catalyzed reactions
Balaced Chemical Equation – lists the initial chemical species
(substrates) present and the new chemical species (products)
formed for a particular chemical reaction
A+BP+Q
The double arrows indicate reversibility in the chemical
reaction, an intrinsic property of all reactions. Unidirectional
arrows are also used to describe reactions in living cells
wherein the products of reaction are immediately consumed
by a subsequent enzyme-catalyzed reaction.
Gibbs Free Energy Change – describes both the direction in
which a chemical reaction will tend to proceed, and the
concentration of reactants and products that will be present at
equilibrium.
 G for a chemical reaction equals the sum of the free
energies of formation of the reaction products minus
the sum of the free energies of formation of the
substrates
 Spontaneous Reactions – the free energy of the
products is lower than that of the substrates,
indicating that the reaction as written is favored in
the direction from left to right
G0 = -ln Keq
o This illustrates the relationship between the
equilibrium constant Keq and G0 , where R is the
gas constant andT is the absolute temperature in
degrees Kelvin
o Keq is equal to the product of the concentration
of the reaction products, divided by the product
of the substrates
Transition State – this is fundamental to understanding the
chemical and thermodynamic basis of catalysis. It is the
transient intermediate where neither free substrate nor
product exists
Activation Energy ( Gf) – The energy required to surmount a
given energy barrier; this is the energy for the reaction
Transcribers: Aclan, J.V., Advento, V., Bolos, C.,Cabiscuelas, Y.N., Cuaderno, C., Gamboa, K.A., Javier, K., Laurilla, L.B., Rodenas, E., Roxas, F.
proceeding in the opposite direction to that draw, which is
equal to -GD.
NUMEROUS FACTORS AFFECT THE FF:
1. The Reaction Rate
a. Kinetic Theory/Collision Theory – for two molecules to
react, they must:
i. Approach within bond forming distance of one
another or collide
ii. Possess sufficient kinetic energy to overcome the
energy barrier for reaching the transition state
 Anything which increases the frequency or energy of
collision between substrates will increase the rate of the
reaction in which they participate
b. Temperature – raising the temperature increases the
kinetic energy of molecules
c. Equilibrium constant Keq – the ratio of k1 to k-1
IMPORTANT PROPERTIES OF A SYSTEMATIC EQUILIBRIUM
A. The equilibrium constant is a ratio of the reaction rate
constants (not the reaction rates)
B. At equilibrium, the reaction rate (not the constants) of
the forward ad back reactions are equal
C. Equilibrium is a dynamic state. Individual substrate and
product molecules are continually being
interconverted
D. The numeric value of the equilibrium constant Keq can
be calculated either from the concentrations of
substrates and products at equilibrium or from the
ratio of k1/k-1
KINETICS OF ENZYMATIC CATALYSIS
 All enzymes accelerate reaction rates by providing
transition states with lowered Gf for formation of the
transitions states.
 The presence of an enzyme therefore has no effect on G0
 Enzymes, therefore, have no effect on Keq
KINETICS
Kinetics is the study of the rate of change of reactants to
products. Velocity expresses the change in the concentration of
substrate per unit time.
For the reaction A  P, the initial velocity is the change in
reactant or product concentration during the first few seconds
of the reaction. Mathematically, the velocity is expressed as:
𝒅𝑨 𝒅𝑷
Velocity (V) = =
𝒅𝒕 𝒅𝒕
Determination of the velocity of a reaction reveals nothing
about the stoichiometry of the reactants and products or about
the reaction mechanism. An equation is needed that relates the
initial velocity to the concentration of the reactants. This is the
RATE EQUATION. In the reaction A -> P the rate equation is:
𝒅𝑨
= V = k[An]
𝒅𝒕
Thus, the initial velocity depends on the starting concentration
of A to the nth power multiplied by a proportionality constant (k)
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 BIOCHEMISTRY Cell and Cell Membrane: A Biochemical Approach

or RATE CONSTANT. The exponent n is usually an integer from 1 -hypothesis that the rate-limiting step in enzymatic reactions is
to 3. the breakdown of the ES complex to product and free enzyme.
This illustrates the mathematical relationship between initial
ORDER OF REACTION
reaction in velocity Vi and substrate concentration (S).
Empirically, the order of a reaction is determined as the sum of
the exponents on each concentration term in the rate 𝑉𝑚𝑎𝑥 [𝑆]
𝑉𝑖 =
expression. 𝐾𝑚+[𝑆]

In A  P, the reaction is FIRST ORDER since the velocity *The derivation starts from the steps of formation and
depends on the concentration of A only. breakdown of ES. Vi is determined by the breakdown of ES to
form product.
In the reaction A + B  C i.e., v = k[A][B], the reaction is
SECOND ORDER. k1 k3
E+S  ES  E+P where E= enzyme
Note that the order of reaction is independent of the   S= substrate
stoichiometry, in a THIRD ORDER reaction, the rate expression k2 k4 P= product
could be either v = k[A][B]2 or v = k[A]2[B]. k1k2k3k4= specific rate constant
for the reactions designated
If the differential first order rate expression v = k[A] is
integrated, we have: Ex. If k3 is very small, Vi =k3 (ES)
where Vi = initial velocity
𝑨
K1 * t = 2.3 log
𝑨−𝑷
1. When substrate concentration is so high and max
Where [A] is the initial reactant concentration and [P] is the velocity is needed, then
concentration of the product formed at time t. The first order Vmax= k3(Et) or Vmax= k3(E) where Et is total bound
rate constant k1 has the units of reciprocal time. enzyme (excess substrate)
E is the total enzyme
Many biological processes proceed under first order conditions. concentration
The clearance of many drugs from the blood by peripheral 2. When (S) is < Km:
𝑉𝑚𝑎𝑥 (𝑆) 𝑉𝑚𝑎𝑥(𝑆) 𝑉𝑚𝑎𝑥
tissues is a first order process. 𝑉𝑖 = 𝑉𝑖 = = ( ) (𝑆) The term
𝐾𝑚+(𝑆) 𝐾𝑚 𝐾𝑚
Km+(S) is equal to Km
A specialized form of the rate equation can be used in these
cases. If we define t1/2 as the time required for the The initial rate of formation is equal to the rate of breakdown
concentration of the reactants or the blood level of a drug to of ES. This is called the steady-state assumption.
be reduced by on-half the initial value, then, the differential k1(E-ES)(S) = k2(ES)+ k3(ES)
first order expression can be simplified to:
(E-ES)(S) + k2+k3 = Km
𝟏
K1 * t1/2 = 2.3 log = 2.3 log 2 (ES) k1
𝟏−(𝟏/𝟐)

𝟎.𝟔𝟗 where Km is the Michaelis Constant

t1/2 =
𝒌𝟏
The Michaelis Constant is the substrate conc. at which V i is half
Many second order reactions that involve water or any one of the maximal velocity (Vmax/2).
the reactants in large excess can be treated as pseudo-first
order reactions. In the hydrolysis of an ester. Rate of Formation of ES can be expressed as:
𝑑(𝐸𝑆)
= 𝑘1(𝐸 − 𝐸𝑆)(𝑆)
The second rate expression is: v=k2 [ester][H2O] but since 𝑑𝑡
water is in abundance, the system obeys the first order rate
law. Reactions in the cell that involve hydration, dehydration, Rate of Breakdown of ES:
–𝑑(𝐸𝑆)
or hydrolysis are pseudo-first-order. = 𝑘2(𝐸𝑆) + 𝑘3(𝐸𝑆)
𝑑𝑡

Is there such a thing as Zero Order? Eto yung pag wala ka

nang pera, Zero Order mo! Significance of Km
Remember that Vi= ½ Vmax. Km=(S) as follows:
The rate expression for the zero order reaction is v=k0. 1 𝑉𝑚𝑎𝑥(𝑆)
𝑉𝑚𝑎𝑥 =
2 𝐾𝑚+(𝑆)

Zero order reaction condition only occurs in catalyzed reactions

2 𝑉𝑚𝑎𝑥 (𝑆)
where the concentration of reactants is large enough to Km+(S) =
𝑉𝑚𝑎𝑥
saturate all the catalytic sites.
Km= S
 Michaelis-Menten Equation Km values are near concentrations of substrate found in cell.
The direct measurement of Vmax often requires high
concentrations of substrate to achieve saturating conditions. A

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 BIOCHEMISTRY Cell and Cell Membrane: A Biochemical Approach

linear form of Michaelis-Menten equation permits Vmax and Km divided into ordered and random). There are many variations
to assume the new value needed for saturation. on these major mechanisms.

𝑉𝑚𝑎𝑥 [𝑆] 1 𝐾𝑚+(𝑆) 1 𝐾𝑚

𝑉𝑖 = invert = factor = +
𝐾𝑚+[𝑆] 𝑣𝑖 𝑉𝑚𝑎𝑥(𝑆) 𝑉𝑖 𝑉𝑚𝑎𝑥(𝑆)
*** “Bi-Bi” reactions- two-substrate, two-product reactions
(𝑆) 𝟏 𝑲𝒎 𝟏 𝟏
simplify = ( ) (𝑺) + 1. Sequential or Single Displacement Reactions
𝑉𝑚𝑎𝑥(𝑆) 𝑽𝒊 𝑽𝒎𝒂𝒙 𝑽𝒎𝒂𝒙
- both substrates must combine with the enzyme to form a
This form is called the Lineweaver-Burk equation or the ternary complex before catalysis can proceed.
double-reciprocal plot. A plot of 1/Vi versus 1/(S) (1/Vi) as y - group undergoing transfer is usually passed directly, in a single
and 1/(S) as x) and whose slope is Km/Vmax will result to a step, from one substrate to the other.
straight line (y= mx+b). This is useful in distinguishing between
different enzymatic reaction mechanisms and enzyme inhibition. a. Random order - either substrate may combine first with the
enzyme to form a complex.
Three types of Inhibition b. Compulsory order - substrate A must bind before substrate B
.
Types of Characteristics Effect on Effect on
Inhibition of the Vmax Km (αKm) In a sequential mechanism, if two substances A and B can bind
Inhibition in any order, it is random mechanism; if binding of A is required
Competitive Binds to the Vmax is the Km is before B can be bound, then it is an ordered mechanism. In
active site same. different. either case, the reaction is bimolecular, i.e., both A and B must
More Km be bound before reaction occurs. Examples are found among
substrate is increases the dehydrogenases in which the second substrate is a
needed to as the coenzyme. Release of products may or may not be ordered in
get half number of either case.
Vmax. inhibitor
increases.

Non- Binds at Vmax Km is the

competitive different sites decreases same
on the enzyme in the
not on the presence of
active site inhibitor
Uncompetitive Only binds to Vmax Km
the ES complex decrease to decrease to
away from the the same the same - Both substrates must combine with the enzyme to form a
active site value as Km values as ternary complex before catalysis can proceed. Sequential
Vmax reactions are sometimes referred to as single-displacement
reactions because the group undergoing transfer is usually
passed directly, in a single step, from one substrate to the
other. ***Sequential Bi-Bi reactions can be further
distinguished based on whether the two substrates add in a
random or in a compulsory order. For random-order
reactions, either substrate A or substrate B may combine
first with the enzyme to form an EA or an EB complex. For
compulsory-order reactions, A must first combine with E
before B can combine with the EA complex (e.g.
Dehydrogenases). One explanation for a compulsory-order
mechanism is that the addition of A induces a
conformational change in the enzyme that aligns residues
1 𝛼𝐾𝑚 1 1
a. 𝑉𝑖 = (𝑉𝑚𝑎𝑥) (𝑆)
+
𝑉𝑚𝑎𝑥 that recognize and bind B. Figure shows: a) ordered b)
b.
1
= (
𝐾𝑚
)
1
+
𝛼𝐾𝑚 random sequential mechanisms.
𝑉𝑖 𝑉𝑚𝑎𝑥 (𝑆) 𝑉𝑚𝑎𝑥
1 𝛼𝐾𝑚 1 𝛼𝐾𝑚
c. 𝑉𝑖
= ( )
𝑉𝑚𝑎𝑥 (𝑆)
+
𝑉𝑚𝑎𝑥
Inhibition occurs as the product progressively inhibits the
reaction as concentration of product increases.

MULTISUBSTRATE REACTIONS

- Most enzymes utilize more than one substrate, or act upon

one substrate plus a coenzyme and generate one or more
products.
- Mechanistically, enzyme reactions are divided into two major
categories: ping-pong and sequential (sequential is further

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 BIOCHEMISTRY Cell and Cell Membrane: A Biochemical Approach

2. Ping-pong Bi-Bi reactions/double displacement reactions -refers to proton transfers mediated by other
- one or more products are released from the enzyme before all classes of molecules
the substrates have been added.

- The ping-pong mechanism can be represented as follows:

ex: Transamination
*AA1 --> give amino group to AKA1
*AKA2 --> will be converted into AA2
(A good example of this mechanism is the transaminase-
catalized reaction in which the alpha amino group of a.a1 is
transferred to the enzyme and the newly formed alpha
ketoacid1 is released, as the first product, followed by the
binding of the acceptor alpha-keto acid2 and released of a.a2.)

UNCATALYZED vs. CATALYZED REACTIONS

- In catalyzed reaction - the activation energy is lowered as
compared to uncatalyzed reaction.
- Ultimately, the main point of enzymatic activity is the
lowering of activation energy
Diagram for catalyzed and uncatalyzed reactions is shown:
The active sites of some enzymes contain amino acid functional
groups that can participate in the catalytic process as proton
donors or proton acceptors.
- e.g. Histidine
- protonated form (most important general acid) and
its conjugate base (most important general base) in
physiologic pH

• other acids
- thiol – SH, tyrosine –OH &
epsilon amino group of lysine (see below)

• other bases
- carboxylic acid anion and their conjugate bases of
general acids

2. Substrate strain
- The energy barrier represented by the uncatalyzed curve is a - Substrates assumes the conformation suitable to the
measure of the activation energy, Ea is required for the reaction transition state
to occur. At the apex of the energy barrier is the activated - Mechanism of strain induction
complex known as the transition state, Ts that represents the • substrate energy level is raised
reactants in their activated state. The Ts complex can • bond length and angles resembles the transition state
breakdown to products or go back to reactants. The overall
energy difference between reactants and products is the same 3. Covalent catalysis
in catalyzed and uncatalyzed reactions. The enzyme-catalyzed - Enzymes (enzyme-bound coenzyme) bond through
reaction proceeds at a faster rate because the energy of covalent bonding
activation is lowered. - Nucleophilic (Nu-) or electrophilic (E+) group attacking the
- In general, enzymatic rate enhancement can be accounted for active site of the enzyme results to covalent bonding of
by the following mechanisms: acid-base catalysis, substrate substrate to the enzyme as an intermediate in the rxn
strain (transition state stabilization), covalent catalysis, ground sequence
state destabilization and entropy effects. A given enzyme may
utilize one or more of these mechanisms.

MECHANISMS OF CATALYSIS
-means that enzymes use in order to overcome a potential
energy barrier to convert reactants to products:

1. Acid base catalysis

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Transcribers: Aclan, J.V., Advento, V., Bolos, C.,Cabiscuelas, Y.N., Cuaderno, C., Gamboa, K.A., Javier, K., Laurilla, L.B., Rodenas, E., Roxas, F.
 BIOCHEMISTRY Cell and Cell Membrane: A Biochemical Approach

Regulation of Enzymatic Activity

Control of a pathway occurs through modulation of the activity

of one or more key enzymes.

Key Enzymes:
1. Rate-Limiting Enzyme
- the enzyme with lowest Vmax; occurs early in the
pathway

2. Enzyme for catalyzing Committed Step

- first irreversible reaction unique to a metabolic
pathway

Note: Rate –Limiting enzyme is not necessarily the enzyme

associated with the Committed Step.

To regulate the activity associated with Rate-Limiting and

4. Transition state stabilization
- Active site binds with the transition state with a much
greater affinity than the substrate
– the substrate will be converted to products FASTER
in a transition state geometry
- Substrate molecules resembling the transition state will
contribute to catalysis (rapid conversion of the substrates
to products)
5. Entropy effect
- Proper orientation and nearness of the substrate with
respect to catalytic groups – INCREASES the rate of
enhancement in enzymes
(“PROXIMITY EFFECT”)
- Decreases entropy
- Increases orderliness associated to the proper
orientation of the substrate
6. Ground destabilization
- Energy barrier can be lowered – raising the ground state
energy
- Destabilization can be achieved by being selective on the:
• Conformation of substrates in the TS
• Enzyme active site residue in the TS
(TS=Transition state)
- Destabilization of ground state = lowering of energy of
activation
Environmental Parameters Influencing Catalytic Activity
- External parameters affect the activity of the enzyme
- May be insignificant under normal conditions but important in
enzyme assays (ex. Samples of patient’s plasma or tissue)
1. Temperature
• High temperature= high speed of reaction until optimum
temperature is reached
= ACTIVITY will eventually DECREASE
• Bell-shaped curve (velocity vs. temperature)
o phosphorylated and dephosphorylated proteins
• Optimum temperature= 40 – 45 C
2. pH
• nearly all enzymes has Bell-shaped pH – velocity profile
• maximum pH varies with different enzymes
Committed Step:
 the absolute amount of the enzyme can be regulated
by change in de novo synthesis of enzyme
 modulate the enzyme activity by activators, by
inhibitors and by covalent modification
 physically partitioning the pathway from its initial
substrate and by controlling access of the substrateto
the enzymes of the pathway (referred as
Compartmentation)
Note:
 the velocity of a reaction is dependent on the amount
of enzyme present
 more enzymes may be synthesized or existing rates of
synthesis repressed through hormonally instituted
activation of the mechanisms controlling gene
expression
 many rate-controlling enzymes are present in very low
concentrations and have relatively short half-lives; this
will provide a mechanism for effecting much larger
fluctuations in the activity of a pathway than would be
possible by inhibition or activation of existing levels of
enzyme
Modifications of the Existing Enzyme Activity for Short-term
Regulation to occur:
1. Feedback inhibition
- when the key enzyme of the synthetic pathway is
inhibited by the end products resulting in shut
down of the pathway
2. Cross regulation
- feedback on the other pathway occurs; product of
one pathway serves as inhibitor or activator of an
enzyme occurring early in the pathway
3. Reversible Covalent Modification
- interconvertible active and inactive forms are
-
e.g. acetylation-deacetylation
adenylation-deanylation
methylation-demethylation
phosphorylation-dephosphorylation
(most common)

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 BIOCHEMISTRY Cell and Cell Membrane: A Biochemical Approach
More on Regulation of Enzymatic Activity
The catalytic capacity of the rate-limiting reaction in a
metabolic pathway is the product of the concentration of the
enzyme and their catalytic efficiency.
I. Concentration of Enzymes
A. Synthesis
1. Inducers (for inducible enzymes)- e.g.
cytochrome P450, enzymes for urea cycle,
HMG CoA reductase
2. Repressors- by excess metabolites
3. Stimulators- hormones and other
extracellular signals
Note: Inducers and Repressors are involved in cis elements
(specific DNA sequences located upstream of regulated genes)
and trans acting regulatory proteins.
B. Degradation
1. Ubiquitin-Proteasome Pathway
a. Proteasome-with more than 30
polypeptide subunits
b. Ubiquitin- small, 75 residue proteins
c. Ubiquitination- covalent attachment of one
or more ubiquitin molecules to a protein to be
targeted by proteasome; catalyzed by E3
ligase
2. Other methods by which recognition is by
proteolytic enzymes can be achieved:
a. covalent modification
b. binding with allosteric effects or
substrate
c. association with membrane
oligonucleotides or other proteins
Note: a growing body of evidence suggests that dysfunction of
the ubiquitin-proteasome pathway contribute to accumulation
of aberrantly folded protein species characteristic of several
neurodegenerative diseases.
II. Catalytic Efficiency
A. Allosteric Regulation
1. Feedback inhibition thru negative allosteric
effector
a. Kinetics- competitive, partially
competitive and non-competitive
b. Typically inhibit the first committed step
in a biosynthetic pathway
2. Multiple Feedback Loops (Cooperative Feedback
Inhibition)
a. Effect may be strictly additive
b. Effect may be greater individual feedback
loop
3. Through hormones by inducing allosteric second
messenger
a. 1st messenger- hormone molecule (nerve
impulse)
b. 2nd messenger- 3’5’ cAMP
Transcribers: Aclan, J.V., Advento, V., Bolos, C.,Cabiscuelas, Y.N., Cuaderno, C., Gamboa, K.A., Javier, K., Laurilla, L.B., Rodenas, E., Roxas, F.
B. Covalent Modification
1. Reversible- phosphorylation (kinase)-
dephosphorylation (phosphatase)- most common
It is most common because:
 it permits the functional properties of the affected
enzyme to be altered only for as long as it serves a
specific need
 the high charge density of phosphoryl group and their
propensity to form strong salt bridges renders them
potent agents for modifying protein structure and
function
 protein phosphorylation is extremely versatile as:
-it affects enzyme catalytic site (most common)
- it alters enzyme location in the cell
- it alters enzyme susceptibility to degradation
- it alters enzyme responsiveness to allosteric
regulation
- it can increase enzyme catalytic efficiency or
converts it to catalytically inefficient form
2. Irreversible-partial proteolysis
Compartmentation
I. Specific subcellular component
A. Enzyme that degrade proteins and polysaccharides- in
lysosomes
B. Enzyme for fatty acid synthesis- in cytosol
C. Enzyme for fatty acid oxidation- in mitochondria
II. Specialized cell type
III. Substitution of one or more reaction by different
reaction favored thermodynamically in opposite
direction
e.g. phosphofructokinase (glycolytic) is replaced by
gluconeogenic-enzyme fructose 1,6 biphosphatase
IV. Ability of enzyme to discriminate between structurally
similar coenzymes
e.g. NAD---- for ATP synthesis
NADPH----for reductive steps in many pathways
Clinical Application of Enzymes
Measuring plasma enzyme activities is based on the premise
that changes in activities reflect changes that have occurred in a
specific tissue or organ.
Functional Plasma Enzymes
- Enzymes that are specific to plasma and serves a
particular function
- Normally present in plasma in high concentration
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 BIOCHEMISTRY Cell and Cell Membrane: A Biochemical Approach

Non-functional Plasma Enzyme especially those with hyperosmolar coma or

diabetes ketoacidosis.
o Monitoring should be done hourly. With this,
you can reverse the situation as fast as you
could.

ELISA
- Enzyme-linked Immunosorbent Assay
- Makes the determination more specific. Combine
enzyme action with antigen-antibody interaction.
- Antibodies specific to a protein antigen are coupled to
an indicator enzyme to generate a very specific and
sensitive assay. After binding of the enzyme-coupled
- Present in plasma but in very low levels. it serves no antibody to the antigen, the enzyme is used to
functional role in plasma generated a colored product that is measurable and
- Under normal conditions, these enzymes should be whose concentration is related to the amount of
undetectable in the plasma. Certain abnormality antigen in a sample. Usually the enzyme is so-called
occurred if these are detected in the plasma. We use horseradish peroxidase.
non-functional plasma enzyme to detect certain
abnormalities Measurement of isozyme.
- Ex. Creatine phosphokinase, lactic dehydrogenase, - Used diagnostically. The most common mechanism for
hydroxybutyrate dehydrogenase which are being used the formation of isozymes involves the arrangements
to detect myocardial infarction of subunits arising from two different generic loci in
o These enzymes are normally in myocardial cell, different combinations to form the active polymeric
if infarction or injury occurs, these enzymes enzyme.
leak to the plasma. - Those with wide clinical application are lactate
o If these enzymes are present in plasma, with dehydrogenase, creatine kinase and alkaline
the presence of characteristic signs and phosphatise.
symptoms [chest pain, hypotensive],
myocardial infarction is confirmed. - Creatine Phosphokinase
- Plasma concentration of each enzyme varies o Dimer, Composed of two subunit [M, (muscle)
depending on time. subunit and B (Brain) subunit]
o Most useful for the acute process: CPK [if test o It has three combinations:
is done between 1- 2 ½ days]  MM – specific for Skeletal muscle
o CPK levels return to below normal after 72  MB- specific for Cardiac muscle
hours  BB- specific for Brain tissue
o If CPK test is done after 56 hours, it will lead o CPK din a masyadong ginagamit kung ang
to a false negative result. suspected ay heart. Kasi nga hindi specific yun.
o LDH can also be used [up to 9 days] - Lactate dehydrogenase:
o Hydroxybutyrate dehydrogenase can also be o Tetramer and has two subunits [H (heart)
used but this is the least specific among the subunit and M(Muscle) subunit]
three. o It has 5 combinations:
- Isozymes are enzymes of same function but can be  HHHH
found on different cell types.  HHHM
- CPK can be found in heart and in other body parts. So  HHMM
we must choose the one that is found in the heart.  HMMM
 MMMM
Enzymes as reagent
Enzymes as therapeutic agents
- Enzymes can be used for screening test for cholesterol
and triglycerides for few minutes using 10ml of plasma. ENZYME FUNCTION
cholesterol oxidase and lipase are the active Streptokinase (prepared It can clear blood clots in
components of the assay system. The enzymes are from streptococcus) Myocardial Infarction or
immobilized in a bilayer along the necessary buffer even in blood clots in
salts, cofactors or cosubstrates and indicator agents. lower extremities to
- Glucose oxidase [for blood sugar] can be embedded prevent cerebrovascular
on a strip of paper. The strip of paper will change its accident
color through the action of the embedded enzyme.
Plasmin A serine-protease
Blood sugar level can be determined by comparing the
enzyme. It cleaves
color on the strip of paper to a reference standard.
insoluble fibrins in blood
This can be done in just 1 minute.
clot into several soluble
o This is important for those who monitor the
components.
blood sugar of patients with diabetes
Asparaginase For treatment of
leukemia

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Transcribers: Aclan, J.V., Advento, V., Bolos, C.,Cabiscuelas, Y.N., Cuaderno, C., Gamboa, K.A., Javier, K., Laurilla, L.B., Rodenas, E., Roxas, F.
 BIOCHEMISTRY Cell and Cell Membrane: A Biochemical Approach

9. Many rate-controlling enzymes have short half-lives.

Ongoing research on genetic diseases: This gives an effective mechanism which will create
- Ex. G6PD Gluscose 6-phosphate dehydrogenase larger fluctuations in the activity of a pathway.
deficiency. Research on how to replace G6PD. a. 1st statement is correct
b. 2nd statement is correct
REVIEW QUESTIONS c. both statements are correct
1. Loosely bound cofactors are called ____. d. neither of the statement is correct
a. Prosthetics group 10. Interconvertible active and inactive forms are
b. Coenzymes phosphorylated and dephosphorylated proteins.
c. Isozymes a. cross regulation
d. Holoenzymes b. allosteric regulation
2. Holoenzymes are c. feedback inhibition
a. Isozyme with cofactor d. reversible covalent modification
b. Apoenzyme with substrate 11. Form of Histidine, which is the most important general
c. Apoenzyme with cofactor base in physiologic pH
d. Isozyme with substrate a. Protonated form
3. This states that the rate of formation of enzyme- b. Conjugate base
substrate complex is equal to the rate of breakdown. c. Both A and B
a. Michaelis- Menten equation d. NOTA
b. Lineweaver- Burk 12. Optimum temperature in catalytic reactions of
o
c. Double reciprocal enzymes is observed between 30-35 C. On the other
d. Steady-state assumption hand, nearly all enzymes shows Bell-shaped pH-
4. Type of inhibition where Km increases as the number velocity profile.
of inhibitor increases. a. Statement 1 is correct
a. Non- competitive b. Statement 2 is correct
b. Competitive c. Both Statements are incorrect
c. Uncompetitive d. Both Statements are correct
d. Mixed inhibition 13. Enzyme being used to clear blood clots in the lower
5. Alpha amino group of alpha-a1 is transferred to the extremities to prevent stroke.
enzyme and the newly formed alpha ketoacid1 is a. Plasmin
released, as the first product, followed by the binding b. Asparaginase
of the acceptor alpha-keto acid2 and released of alpha- c. LDH
a2. This enzymatic reaction is best describe as: d. Streptokinase
a. Sequential reaction 14. Lactate Dehydrogenase is a ________ with ______
b. Transamination possible combinations.
c. Ping-pong reaction a. Tetramer, three
d. Both A and B b. Tetramer, five
e. Both B and C c. Dimer, three
6. Multisubstrate enzymatic reaction wherein either d. Dimer, five
substrate may combine first with the enzyme to form a 15. Group of enzymes that catalyze the addition or
complex. removal of water, ammnonia or carbon dioxide to
a. Sequential; Compulsory order double bonds.
b. Sequential; Random Order a. Hydrolases
c. Ping-pong reaction b. Isomerases
d. Double displacement reaction c. Ligases
e. Both C and D d. Lyases
7. Evaluate the two statements: e. Oxidoreductases
A: The overall energy difference between reactants
and products is the same in catalyzed and uncatalyzed
reactions.
B: The enzyme-catalyzed reaction proceeds at a slower
rate because the energy of activation is increased.
a. Only A is correct ANSWERS TO REVIEW QUESTIONS
b. Only B is correct
15.
14.
13.
12.
11.
10.
9.
8.
7.
6.
5.
4.
3.
2.
1.

c. Both statements are correct

D
A
D
B
B
D
C
C
C
B
E
B
D
C
B

d. Neither of the statement is correct.

8. Recognition by proteolytic enzymes cannot be
achieved by:
a. covalent modification
b. binding with allosteric effects or substrate
c. release of second messenger
d. association with membrane oligonucleotides or
other proteins

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Transcribers: Aclan, J.V., Advento, V., Bolos, C.,Cabiscuelas, Y.N., Cuaderno, C., Gamboa, K.A., Javier, K., Laurilla, L.B., Rodenas, E., Roxas, F.