1.

04
30 June 2015 Dr. Menorca | Enzymes
Enzymes

 Biological polymers that act to catalyze chemical reactions

(substrate  products)
 Possesses substrate specificity and stereospecificity
 Increases rate of chemical reaction by a factor of at least
106
 Neither consumed nor permanently altered
 Temporarily covalently bonded to a molecule
 ONLY increase the rate by which a reaction will reach
equilibrium
 Selective catalyst-specific for a substrate or small set of
related substrates
 Stereospecific - catalyze only one isomer of a compound

Rate/Velocity Substrate
 speed of a chemical reaction  Molecule acted upon by the enzyme to form the product
 Rate - change in the amount (moles) of starting materials  For reversible reactions, products of the forward reaction
or products per unit time become substrate for the reverse reaction
 Velocity - change in the conc.of starting materials or
products per unit time Substrate Binding Site
 Particular region of the enzyme surface where specificity
Apoenzyme resides
 Protein part of an enzyme without any cofactors or
prosthetic groups Active Site
 Inactive enzyme  Found in the substrate binding site
 Contiguous to the substrate binding site in primary
Prostethic Groups sequence or lie in the distal regions of the primary
 tight, stable incorporation into a protein’s structure by sequence
covalent or noncovalent forces  Brought adjacent to the substrate-binding site by folding in
 metal ions - metalloenzymes the tertiary structure

Cofactors Allosteric site

 Organic or inorganic molecules  Region in the enzyme that is not at the active site or
 Required by apoenzyme for its activity substrate binding site
 Usually metal ions  Unique site where small molecules bind and affect a
- Metallo-activated enzymes change in the substrate binding site or active site
 Tightly bound cofactors to an apoenzyme are called
prosthetic group
 Loosely bound cofactors are called coenzymes

Holoenzymes
 Apoenzyme together with its cofactor
 Complete and catalytically active

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pagatpatan Page 1 of 11
 BIOCHEMISTRY
1.04 Dr. Menorca | Enzymes

6 CLASSES

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 cleavage of C—C, C—O, C—N, and
otherbonds by atom elimination, leaving double
bonds)
5. Isomerases
- (catalyze geometric or structural changes within a
molecule)
6. Ligases
Isozyme
- (catalyze the joining together of two molecules
 Enzymes variants that catalyze the same chemical reaction
coupled to the hydrolysis of ATP)
Coenzyme
Enzyme Kinetics
 recyclable shuttles
 Group transfer agents that transport many substrates from  study of the rate of reactants to products
their point of generation to their point of utilization.
 In the reaction:
Classification
Change of A to P where:
based on reaction catalyzed, followed by suffix –ase
A = substrate P = product
Examples:
 The initial velocity is the change in reactant or product
 Dehydrogenases remove hydrogen atoms.
concentration during the first few seconds of the reaction.
 Proteases hydrolyze proteins. - If we express the velocity in which A changes to P,
 Isomerases catalyze rearrangements in configuration mathematically, it is expressed as:

Modifiers may precede the name to indicate:

 The substrate, e.g. xanthine oxidase
 The source of enzyme, e.g. pancreatic ribinuclease
 It regulation, e.g. hormone-sensitive lipase  More importantly, we shall express this as a rate equation:
 Feature of its mechanism of action e.g. cysteine protease

Alphanumeric designators are added to identify multiple forms

of an enzyme. Examples are RNA polymerase IIII and protein
kinase Cβ  Rate equation – relates the initial velocity to the
concentration of the reactants d = derivative
IUB - This means that the initial velocity depends on the
 International Union of Biochemistry starting concentration of A raised to the nth power
 developed an unambiguous system of enzyme multiplied by a proportionality constant (k) or Rate
nomenclature in which each enzyme has a unique name constant
and code number that identify the type of reaction - The exponent n is usually an integer from 1 to 3
catalyzed and the substrates involved

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pahatpatan Page 2 of 11
 BIOCHEMISTRY
1.04 Dr. Menorca | Enzymes

Order of Reaction
Other Orders of Reactions:
- The sum of the exponents on each concentration term Differential Rate
in the rate expression. Remarks
Expression
2nd
First-Order Reaction for A+B  P:
Order Unit of k is 1/conc∙time
 Velocity depends on the concentration of A: v=k[A][B]
AP
3rd v=k[A]2[B] or
Order v=k[A][B]2
 Differential Rate Expression: v = k[A]; which, upon
integration, yields:

Number of enzyme molecules

is limited in relation to
[A] = Initial reactant concentration substrate molecules
[P] = concentration of the product formed at time t
Zero Order v=k0
K1 = first order rate constant; unit: Catalytic sites are therefore
saturated
 Many biological processes proceed under 1st order
Unit of k is concentration/time
conditions. The metabolism of many drugs from the blood
by peripheral tissues is a 1st order process.
 The time required is usually measured in t½ (half-life).
- If t½ is used to represent the time required for the
concentration of the reactants or the blood level of a These are 2nd order reactions that involve water or
drug to be reduced by half the initial value, a simplified any reactant in large excess. Eg, in the hydrolysis of
equation can be obtained: ester:

Pseudo- R-C-O-CH3 + H2O  R-C-OH + CH3OH

1st
½ Order This should be a 2nd order reaction with
Transmutation will yield, v=k2[ester][H2O], but since water is in abundance,
First-Order Half-Life Equation: the system obeys the 1st order rate law.
Reactions in the cell that involve hydration,
dehydration, or hydrolysis are pseudo-first order.

Eg: A certain 10-mg drug is metabolized via a 1st-order process Michaelis-Menten Equation
& has a rate constant of 2.87x10-2 hr-1. How long will it take for Given the Reaction Model:
the drug to reach a 25% concentration in the plasma?

Answer: 48 hrs

Where: E = enzyme; S = substrate; ES = Enzyme-substrate

24 hrs ½
complex; P = product; k1 and k3 are rate constants for the
(Drug is at 50% blood level by the 24th hr; and by extension, will
forward reactions; k2 and k4 are rate constants for the backward
achieve half of that ie, 25% concentration, after another 24 hrs)
reactions.

 Since [ES] is hard to measure experimentally, total enzyme

concentration, [Et], will be used; and [Et]-[ES] to represent
free/unbound enzyme.
 Initial velocity (Vi) is determined by the rate-limiting step of
enzymatic catalysis: the breakdown of ES to form the
product P.
vi = k3[ES]

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pahatpatan Page 3 of 11
 BIOCHEMISTRY
1.04 Dr. Menorca | Enzymes

 Maximum velocity (Vmax) occurs when the enzyme is

saturated (that is, when [Et]=[ES]). Thus, it can be expressed Variations on the Michaelis-Menten Equation:
as:
Vmax = k3[Et]

Derivation Steps:
1. Rate of formation and breakdown of ES will be represented
by the following equations, respectively:

Rate of ES formation = k1[Et-ES][S]

Rate of ES breakdown = k2[ES]+k3[ES]

2. The steady-state assumption allows us to assume that rate

of ES formation = rate of ES breakdown; this assumption
states that the initial rate of reaction reflects a steady state
in which [ES] is constant. k1[Et-ES][S] = k2[ES]+k3[ES]
1. When [S] is too low compared to Km
3. From here, we can compute for [ES] algebraically, which - The [S] in the denominator of the equation becomes
will yield: negligible
- New equation:

- Graphical representation: Linear dependence of vi to

*wherein Km is the Michaelis constant, which is a
[S]
simplification of the part of the
equation. 2. When [S] = Km
*the Michaelis constant represents the substrate - vi becomes half-maximal
concentration at which vi is half the maximal velocity - New equation:
(Vmax/2) attainable at a particular concentration of
the enzyme.
3. When [S] is too high compared to Km
4. Incorporating vi = k[E][ES] = k3[ES] into the equation: - The Km in the denominator becomes negligible
- New equation:

5. Incorporating Vmax = k3[Et] into the equation: - Graphical representation: Plateau observed at high [S]

Lineweaver-Burk Plot

- A double-reciprocal variation of the Michaelis-Menten

equation which yields a linear graph; makes it easier to
measure direct value of Vmax and Km through extrapolation.
This is the Michaelis-Menten Equation. It illustrates in
mathematical terms the relationship between initial reaction
velocity vi and substrate concentration [S].
Plotting will yield a hyperbolic graph.
y-axis is 1/v
Where: x-axis is 1/[S]
Km = Michaelis constant y-intercept is 1/Vmax
Vmax = Maximal velocity slope is Km/Vmax
vi = initial reaction velocity
[S] = substrate concentration

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pahatpatan Page 4 of 11
 BIOCHEMISTRY
1.04 Dr. Menorca | Enzymes

Types of Enzyme Inhibition Compulsory-Order

 substrate A must first combine with enzyme E before
1. Competitive Inhibitors - decrease the number of free enzyme substrate B can combine with EA (enzyme-substrate A
molecules available to bind substrate; can be overcome by complex)
increasing [S]  addition of substrate A induces a conformational change in
Vmax: no change the enzyme E that aligns residues that recognize and bind
Km: ↑ substrate B
 in a linear presentation
2. Non-Competitive Inhibitors - Bind enzymes at sites distinct  characteristic of many NAD(P)H-dependent
from the substrate-binding site; binding of the inhibitor does oxidoreductases
not affect binding of the substrate.  “Bi Uni Uni Bi”

Vmax: ↓
Km: no change

3. Uncompetitive Inhibitors – Bind to the ES complex in the

presence of a substrate. E – enzyme
P – first product
Vmax: ↓ (same magnitude w/ Km) A – substrate A
Km: ↓ (same magnitude w/ Vmax) Q – second product
B- substrate B

Random-Order
 either substrate A or substrate B may combine first with
the enzyme to form an EA or EB complex
 characteristic of many kinases and some dehydrogenases
 “Bi Uni Bi Uni”
Multi-substrate Reactions
*From Harper and Lehninger 

 involve transfer of an atom or a functional group from one

substrate to the other

Ping-Pong or Double-Displacement Reactions

Sequential or Single-Displacement Reactions

 both substrates must combine with enzyme to form a non-
covalent ternary complex before catalysis can proceed
 group undergoing transfer is passed directly (in a single
step) from one substrate to the other
 one or more products are released from the enzyme before
Sequential Bi-Bi reactions all the substrates have been added
1. Compulsory-Order  involves covalent catalysis and transient, modified form of
2. Random-Order the enzyme
 characteristic of aminotransferases and serine proteases

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pahatpatan Page 5 of 11
 BIOCHEMISTRY
1.04 Dr. Menorca | Enzymes

 first substrate is converted to product and dissociates Catalyzed vs Uncatalyzed Reactions

before the second substrate binds  no ternary complex is
formed
 substrate 1 may transfer a functional group to the enzyme
(to form the covalently modified E’) which is subsequently
transferred to substrate 2
Steady–state Kinetics
 determines whether a ternary complex is formed during
the reaction
 direct measurement of the rates of individual reaction
steps (association of enzyme and substrate to form the ES
complex)
 show how energy changes during the reaction and it is used
by a specific enzyme
 enzymes accelerate reaction rates by lowering ΔGF
(activation energy) for the formation of transition states
 the environment of the active site lowers ΔGF by stabilizing
the transition state intermediates
involve:
- acid-base groups suitably positioned to transfer
protons to or from the developing transition state
intermediate;
- charged groups or metal ions that stabilize developing
charges; and
- imposition of the steric strain on substrates so that
their geometry approaches that of the transition state.

Mechanisms of Catalysis

Enzymes can enhance the rates of reaction by a factor of 10 9–

1012 times that of the noncatalyzed reaction

1. ACID-BASE CATALYSIS
 The ionizable function group of the aminoacyl side chains
and (where present) of prosthetic groups contribute to
catalysis by acting as acids or bases.
For (a) and (b):  Can either be specific or general
 [substrate 1] – varied o [substrate 2] – constant; repeated - Specific Acid-Base Catalysis – the rate of reaction is
for several values, generating several separate lines sensitive to changes in the concentration of protons
 intersecting lines  a ternary complex is formed but independent of the concentrations of other acids
 parallel lines  Ping-Pong pathway (proton donors) or bases (proton acceptors) present in
solution or at the active site.
Product inhibition studies - specific acids: H+ or H30+ ; bases: OH-
 distinguish between ordered and random Bi-Bi reactions - General Acid-Base catalysis – reaction rates are
- random-order - each product will be a competitive responsive to all the acids or bases present; proton
inhibitor regardless of which substrate is designated transfers mediated by other classes of molecule;
the variable substrate occurs in vast majority of enzymes
- compulsory-order - only the product can give the - general acid/base – weakly ionizable
pattern whether competitive or non-competitive compound
inhibition had occurred - ie, active site of Ribonuclease (RNAse)

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pahatpatan Page 6 of 11
 BIOCHEMISTRY
1.04 Dr. Menorca | Enzymes

 Common among enzymes that catalyze group transfer

reactions
- Residues on enzymes: cysteine or serine, occasionally
histidine
 often follows a “ping pong” mechanism – the first substrate
is bound and its product released prior to the binding of
the second substrate
 i.e. serine proteases (trypsin, chymotrypsin, thrombin)

The active sites of some enzymes contain amino acid functional

groups that can participate in the catalytic process as proton 4. TRANSITION STATE STABILIZATION
donors or proton acceptors.  active site binds the transition state (TS) with a much
greater affinity than the substrate
2. SUBSTRATE STRAIN  mass action: all substrate converted rapidly to products
 Enzymes that catalyze –lytic reactions (break covalent  any factor that increases the population of substrate
bonds) bind their substrates in a conformation (mimics the molecules resembling the TS will
*transition state intermediate) slightly unfavorable for the  contribute to catalysis
bond that will undergo cleavage.
 The resulting strain distorts the targeted bond  weak 5. ENTROPY EFFECT
substrate  vulnerable to cleavage  Entropy (S): extent of disorder in a system
 i.e. mechanism of lysozyme action  At equilibrium, entropy is maximal
- transient species representing half-way point in the  Decrease in entropy contributes to the rate of reaction by a
transformation of substrates to products factor of 103
 An enzyme with high affinity binding sites for substrates
will cause the substrates to bind with the enzyme in the
correct orientation  effective concentration of reactants
will
 increase  increased reaction rate

Environmental Parameters Influencing Catalysis

 External parameters affecting enzyme activity
 May be insignificant in vivo under normal conditions, but
are very important in in vitro enzyme assays (e.g.
 Samples of patient’s plasma or tissue)

1. Temperature
 Plot of Velocity vs Temperature reveals Bell-shaped curve
3. COVALENT CATALYSIS (optimum temp: 40-45°C)
 formation of a transient covalent bond between the  High temperature = High speed of Reaction (will eventually
enzyme and substrate due to attack of nucleophilic decrease)
(negatively charged) or electrophilic (positively charged)
group in the enzyme active site 2. pH
 the modified enzyme becomes a reactant  nearly all enzymes show a bell-shaped pH-velocity profile
 introduces a new reaction pathway: activation energy is  initial increase in velocity, peak(max), then decrease in
lower and therefore faster velocity
 completion of the reaction causes the enzyme to return to  maximum velocity varies greatly with different enzymes
its original unmodified state
3. Other:
- Salt Concentration

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pahatpatan Page 7 of 11
 BIOCHEMISTRY
1.04 Dr. Menorca | Enzymes

4. Transition State Stabilization More on Regulation

5. Entropy Effect
6. Ground Destabilization I. Concentration of enzymes
 Lowering the energy barrier by raising the energy of the  Regulation of synthesis
ground state - A1. Inducer (e.g. cytochrome p450, enzymes for urea
 Can be accomplished by Selective cycle, HMG coA reductase)
 Conformation of substrates in TS; - Enzyme active-sites - A2. Repressors – by excess metabolites
residues in TS (TS=Transition State) - A3. Stimulators – hormones and other extracellular
signals
Regulation of Enzyme Activity  Regulation of degradation
- B1. Ubiquitin proteasome pathway – proteasome is a
 Control of pathway occurs through modulation of the 30-polypeptide subunit protein that degrades protein
activity of one or more key enzymes in the pathway. with attached ubiquitin. Ubiquitination = covalent
 Rate Limiting Enzyme- enzyme with the lowest Vmax. attachment of ubiquitin to proteins to be degraded by
 Committed Step- is the first irreversible reaction that is proteasome
unique to a metabolic pathway. - (there’s a growing body of evidence which suggest
 The activity of the enzyme can be regulated by: that a dysfunction in this pathway contribute to
1. The absolute amount of the enzyme can be regulated the accumulation of aberrantly folded protein
in by change in the de novo synthesis of the enzyme. species which are characteristic of some
2. The activity can be modulated by activators, inhibitors neurodegenerative diseases)
and covalent modification. - B2. Other methods by which proteolytic enzymes
3. By physically partitioning the pathway from its initial recognize enzymes to be degraded:
substrate and by controlling the access of the substrate - covalent modification; binding with substrate or
to the enzymes of the pathway (compartmentation) . allosteric effects; and association with
- The velocity of any reaction is dependent on the oligonucleotides or other proteins
amount of enzyme present.
- Many rate controlling enzymes have relatively II. Catalytic efficiency
short half-lives.  Allosteric regulation
1. Feedback inhibition thru negative allosteric effector
SHORT-TERM REGULATION a. Kinetics -competitive, partially competitive
 Short-term regulation occurs thru modification of the and non-competitive
activity of existing enzyme. b. Typically inhibit the first committed step in a
 Feedback Inhibition- when the key enzyme of the synthetic biosynthetic pathway
pathway is inhibited by the end products resulting in shut 2. Multiple Feedback Loops (Cooperative Feedback
down of the pathway. Inhibition)
 Cross Regulation- the feedback on the other pathway. A a. Effect may be strictly additive
product of one pathway serves as an inhibitor or activator b. Effect may be greater individual feedback
of an enzyme occurring early in another pathway. loop
 Reversible Covalent Modification- inter convertible active 3. Through hormones by inducing allosteric second
and inactive forms are phosphorylated and messenger
dephosphorylated proteins. a. 1st messenger-hormone molecule (nerve
e.g. impulse)
Acetylation - deacetylation b. 2nd messenger- 3’5’ cAMP
adenylation - deanylation
methylation - demethylation
 Covalent Modification
phosphorylation - dephosphorylation
1. Reversible-phosphorylation
There are enzymes that when phosphorylated become active, (kinase)dephosphorylation
while others become inactive. This depends upon the enzyme. (phosphatase) is most common because:
SO KNOW WHICH ENZYMES ARE ACTIVATED BY  it permits the functional properties of the affected
PHOSPHORYLATION. NOTE: The phosphorylation– enzyme to be altered only for as long as it serves a
dephosphorylation scheme is the most common  specific need
 the high charge density of phosphoryl group and
their propensity to form strong salt bridges

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pahatpatan Page 8 of 11
 BIOCHEMISTRY
1.04 Dr. Menorca | Enzymes

renders them potent agents for modifying protein - If these enzymes are present in plasma, with the
structure and function presence of characteristic signs and symptoms [chest
protein phosphorylation is extremely versatile as: pain, hypotensive], myocardial infarction is confirmed.
o it affects enzyme catalytic site (most - Plasma concentration of each enzyme varies
common) depending on time.
- Isozymes are enzymes of same function but can be
o it alters enzyme location in the cell
found on different cell types.
o it alters enzyme susceptibility to
- CPK can be found in heart and in other body parts. So
degradation
we must choose the one that is found in the heart.
o it alters enzyme responsiveness to
allosteric regulation
Most useful for the acute process
o it can increase enzyme catalytic efficiency
or converts it to catalytically inefficient if test is done between 1-2 ½ days
form CPK levels return to below normal
2. Irreversible – partial proteolysis CPK after 72 hours

III. Compartmentation If CPK test is done after 56 hours,

1. Specific subcellular component it will lead to a false negative
- Enzyme that degrade proteins and result.
polysaccharides- in lysosomes
- Enzyme for fatty acid synthesis- in cytosol LDH can also be used up to 9 days
- Enzyme for fatty acid oxidation -in mitochondria
2. Specialized Cell type
3. Substitution of one or more reaction by different Hydroxybutyrate can also be used but this is the
reaction favored thermodynamically in opposite dehydrogenase least specific among the three
direction
- e.g. phosphofructokinase (glycolytic) is replaced by
gluconeogenic-enzyme fructose-1,6-
bisphosphatase Enzymes as Reagents
4. Ability of enzyme to discriminate between  Enzymes can be used for screening test for cholesterol and
structurally similar coenzymes eg, triglycerides for a few minutes using 10ml of plasma.
- NAD - for ATP synthesis  Cholesterol oxidase and lipase are the active components
- NADPH - for reductive steps in many pathways of the assay system.
 The enzymes are immobilized in a bilayer along the
Plasma Enzymes necessary buffer salts, co-factors or cosubstrates and
indicator agents.
Functional Plasma Enzymes  Glucose oxidase (for blood sugar) can be embedded on a
- Enzymes that are specific to plasma and serves a particular strip of paper. The strip of paper will change its color
function through the action of the embedded enzyme. Blood sugar
- Normally present in plasma in high concentration level can be determined by comparing the color on the
strip of paper to a reference standard. This can be done in
Non-functional Plasma Enzymes just 1 minute.
- Present in plasma but in very low levels  This is important for those who monitor the blood sugar of
- Serves no functional role in plasma patients with diabetes especially those with hyperosmolar
- Under normal conditions, these enzymes should be coma or diabetes ketoacidosis.
undetectable in the plasma  Monitoring should be done hourly. With this, you can
- Certain abnormalities occur if these are detected in the reverse the situation as fast as you could.
plasma
- eg, Creatine phosphokinase (CPK), lactic dehydrogenase Enzyme-Linked Immunosorbent Assay (ELISA)
(LDH), hydroxybutyrate dehydrogenase  Makes the determination more specific.
- Used to detect myocardial infarction  Combine enzyme action with antigen-antibody interaction.
- These enzymes are normally in myocardial cell, if - Antibodies specific to a protein antigen are coupled to
infarction or injury occurs, these enzymes leak to the an indicator enzyme to generate a very specific and
plasma. sensitive assay.
- After binding of the enzyme-coupled antibody to the
antigen, the enzyme is used to generate a colored

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pahatpatan Page 9 of 11
 BIOCHEMISTRY
1.04 Dr. Menorca | Enzymes

product that is measurable and whose concentration is Restriction fragment length polymorphisms (RFLPs)
related to the amount of antigen in a sample. - Deviations in the normal product pattern
- Enzyme is so-called horseradish peroxidase. - occur if a mutation renders a restriction site unrecognizable
to its cognate restriction endonuclease or generates a new
Measurement of Isozyme recognition site
 Used diagnostically. The most common mechanism for the - utilized to facilitate a prenatal detection of hereditary
formation of isozymes involves the arrangements of disorders like sickle cell trait, beta-thalassemia, infant
subunits arising from two different generic loci in different phenylketonuria, and Huntington’s disease
combinations to form the active polymeric enzyme.
- Those with wide clinical application are lactate Polymerase Chain Reaction (PCR)
dehydrogenase, creatine kinase and alkaline - uses a thermostable DNA polymerase and appropriate
phosphatise. oligonucleotide primers to produce thousands of copies of
 Creatine Phosphokinase segment DNA
- Dimer, Composed of two subunit - enables medical, biological, and forensic scientists to detect
- M (muscle) subunit and characterize DNA present initially at levels too low for
- B (Brain) subunit direct detection
- 3 combinations: - used to detect and identify pathogens and parasites such as
- MM – specific for Skeletal muscle Trypanosoma cruzi (causative agent for Chagas disease),
- MB – specific for Cardiac muscle and Neisseria meningitides (causative agent for bacterial
- BB – specific for Brain tissue meningitis)
- CPK di na masyadong ginagamit kung ang suspected ay
heart. Kasi nga hindi specific yun. RECOMBINANT FUSION PROTEINS ARE PURIFIED BY AFFINITY
 Lactate dehydrogenase CHROMATOGRAPHY
- Tetramer and has two subunits - Recombinant DNA technology can be used to create
- H (heart) subunit modified proteins that are readily purified by affinity
- M(Muscle) subunit chromatography
- 5 combinations: - The gene of interest is linked to an oligonucleotide
- HHHH sequence that encodes a carboxyl or amino terminal
- HHHM extension to the encoded protein.
- HHMM - The resulting modified protein, termed a fusion protein,
- HMMM contains a domain tailored to interact with a specific
- MMMM affinity support. One popular approach is to attach an
oligonucleotide that encodes six consecutive histidine
Enzyme as therapeutic agents residues. The expressed “His tag” protein binds to
chromatographic supports that contain an immobilized
divalent metal ion such as Ni2+
Enzyme Function
It can clear blood clots in
Streptokinase (prepared Myocardial Infarction or even in
from streptococcus) blood clots in lower extremities to
prevent cerebrovascular accident.
A serine-protease enzyme. It
cleaves insoluble fibrins in blood
Plasmin
clot into several soluble
components.

Asparaginase For treatment of leukemia.

Ongoing Researches
Restriction endonucleases
- cleave double-stranded DNA at sites specified by a
sequence of four, six, or more base pairs called restriction
sites
- This produces a characteristic set of smaller DNA fragments

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pahatpatan Page 10 of 11
 BIOCHEMISTRY
1.04 Dr. Menorca | Enzymes

The figure illustrates the purification of a GST-fusion protein

using an affinity support containing bound glutathione. Fusion
proteins also often encode a cleavage site for a highly specific
protease such as thrombin in the region that links the two
portions of the protein. This permits removal of the added
fusion domain following affinity purification.

Site-directed mutagenesis
- changes specific aminoacyl residues by altering their
codons.
- Used in combination with kinetic analyses and x-ray
crystallography, this approach facilitates identification of
the specific roles of given aminoacyl residues in substrate
binding and catalysis.
- For example, the inference that a particular aminoacyl
residue functions as a general acid can be tested by
replacing it with an aminoacyl residue incapable of
donating a proton.

Transcribers: De Los Reyes, Fernandez, Flores, Guevarra, Javier, Lanuza, Libit, Maaño, Molina, Pahatpatan Page 11 of 11