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Enzymology
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Index
• Enzymology: Definitions
• Enzyne kinetics
• Calculations for enzyme activity
• Types of enzymatic assays
• Principles of enzyme analysis
• Kinetic colorimetric method
• Kinetic methods based on NADH/NADPH
• Enzymes as reagents
• Hexokinase
• Glucose oxidase
• Technical aspects of enzymatic analysis
• Samples
• Reagents and instruments
• Quality control
• Common enzymatic determinations and its clinical relevance
• Phosphatases (hydrolases-esterases)
• Creatine kinase (CK)
• Transaminases
• Gamma glutamyl transferase
• Amylase
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Enzymology: Definitions
• Enzyme: complex proteins that produce a specific chemical
change on specific substrates in a given cellular compartments.
• Considered as biological catalysts that decrease the activation
energy of a target (and specific) chemical reaction.
Group (EC) Class Catalyzes: Reaction Example
Alcohol
1 Oxidoreductases redox reactions (e- transference) A-+B→A+B-
dehydrogenase
transfer or exchange of certain groups
2 Hydrolaseses A-B+C→A+ B-C Hexokinase
among some substrates
3 Hydrolases hydrolysis of substrates A-B +H2O→ A-H + B-OH Trypsin
removal of a group from the substrate
4 Lyases to leave a double bond reaction or its C-A-B-DA=B + C-D Piruvate decarboxilase
reverse reaction
conversion of geometric or optical
5 Isomerases C-A-B-DD-A-B-D Maleate isomerase
isomers.
synthesis of one compound from
6 Ligases A+B→A-B DNA ligase
substrates with the release of energy
The chain adopts a three-dimensional structure that can be arranged in three different
ways: α-helix, folded β-sheet and spiral (also referred as secondary structure). The
tertiary structure is determined by the ability of the secondary structure to fold back
on itself. Each protein acquires its own tertiary structure that confers the specific
biological properties and activity unique to each enzyme. The polypeptide chains of
the tertiary structure can be joined together, determining the quaternary structure.
These structures determine the catalytic activity of an enzyme and their stability
depends on certain cellular parameters:
- Temperature
- pH
- Ionic strength
The most important region of an enzyme's structure is the so-called active site, which
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is the place where the (specific) substrate binds so that the enzyme can carry out its
catalytic function, the rest of the polypeptide chain being a sort of structural "scaffold"
that guarantees the integrity of the active site.
- They will only affect the rate at which equilibrium is reached in the reaction.
Enzymes are present in all tissues, although not all have the same type of enzymes
nor in equal quantity. Knowledge of their location and function in tissues is what
allows us to use them as markers of certain pathologies.
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Enzyme kinetics
• Enzyme catalyzed reactions occurs by the formation of an
intermediate enzyme-substrate complex:
Kinetics is the study of the rate of enzymatic reaction
and is determined as the rate of product appearance or
substrate disappearance:
E+S→ES→E+P
Actividad: what constant measures specificity of an enzyme for its substrate? What does it represent?
Enzymes working
The way for the enzyme performs its catalytic activity is 1) bind the substrate and 2) to
facilitate the modification of the substrate and 3) release a product. The enzymes
react by offering their active site to the substrate forming the enzyme-substrate
complex, where it acts very quickly until the transformed product is released. The
enzyme is not permanently, "recycling" the enzyme.
Within the whole group of enzymes, there are those that have a high specificity, i.e.,
they only accept one type of molecule on which to perform catalysis. Others, on the
other hand, are less specific and catalyze reactions using molecules with a certain
similarity as substrates.
The interaction between enzyme and substrate takes place through weak bonds
between the substrate molecule and the active site. The greater the number of these
bonds, the greater the specificity of the enzyme.
Enzyme kinetics:
The rate of catalysis of an enzyme is determined either as the rate at which the
product is formed, or as the rate at which the substrate disappears.
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If we keep the reaction conditions constant (pH, temperature, cofactors and enzyme
concentration), the rate increases as we increase the substrate concentration, until
we reach a point (maximum rate) from which the reaction rate is constant even if the
substrate concentration increases. This is explained by the fact that when the amount
of substrate is greatly increased, the enzyme becomes saturated, and it is then that
the highest possible reaction rate is reached, which will depend exclusively on the
time required by the enzyme to transform the substrate.
The Michaelis-Menten model explains the enzymatic activity through the reaction rate
with respect to the substrate concentration.
The Michaelis constant or Km is the substrate concentration in moles/L at which the
reaction rate is half the maximum rate. This constant is characteristic of each pair
enzyme/substrate and allows us to assess the specificity of the enzyme for the
substrate. For a high Km, half of the maximum velocity will be reached at a high
substrate concentration, which means that the enzyme's affinity for that substrate is
low. If the Km of an enzyme for its substrate is low, the affinity of the enzyme for that
substrate will be high, and the velocity fast.
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Calculations for enzyme activity
• Enzyme activity:
• Measured at 37ºC (Standard physiological conditions) and satured:
1 katal= 1 mol of transformed substrate/s
(amount of enzyme that transform 1 mol of substrate per second)
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Types of enzyme assays
• All enzyme assays measure either the consumption of substrate or production of product over time
• Continuous: the progress of the reaction is followed as it occurs. Sometimes referred to as “kinetic assays”.
• Spectrophotometric: measure changes in absorbance of a product or substate (if is in the visible region is colorimetric assay). If
there is no absorbance of substrates or products, it may be necessary to use a coupled reaction.
• Chemiluminescent: measure the light produced during the reaction. Typically use luminol or luciferin
• Stopped: measure how much product is formed over a given time or, in some cases, how much substrate has been used up.
Sometimes referred to as “endpoint assays”.
• Spectrophotometric
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Principles of enzyme assays
• Current methodologies to analyze enzyme activity are:
• Colorimetric kinetic methods:
• Based on observation of substrate/products concentration variation
based on colormetric reaction (using spectrophotometry)
ACP
α-Naphtyl-P + H2O α-Naphtol + Pi
pH 5
α-Naphtol + Fast Red TR Azo dye
30-37ºC
Absorbance
The kit includes L-tartrate to inhibit 405 nm
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Principles of enzyme assays
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Principles of enzyme assays
• Current methodologies to analyze enzyme activity are :
• Colorimetric kinetic methods based on NADH/NADPH:
• Takes advantage of absorbance of coenzymes NADH and NADPH and
not of its oxidized form (NAD+/NADP+)
NAD+/NADP+
Both nucleotides are coenzymes: that is, small non-protein organic molecules that
carry chemical groups between enzymes. They are sometimes called co-
substrates. These molecules are substrates for enzymes and are not a permanent
part of the enzyme structure. This distinguishes coenzymes from prosthetic groups,
which are non-protein components that are tightly bound to enzymes, such as iron-
sulfur centers, flavin or heme groups.
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Principles of enzyme assays
A basal readout is taken at 340 nm. followed by several more readouts at identical
time intervals. The differences between each reading and the previous one defines
the decrease in absorbance and the intervals should be equal throughout the course
of the reaction. At the end of the reaction, the arithmetic means of the differences
found is calculated and multiplied by a factor to obtain the enzyme activity. This factor
by which the increase in absorbance (ΔA) is multiplied is usually included in the
reaction kit specifications and is memorized in the instrument.
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Principles of enzyme assays
We can use enzymes as reagents to determine a given substrate (as target analyte)
in the sample. Thus, to determine the concentration of a substrate, reaction systems
are used in which all the components necessary for it to occur are present except for
the substrate which is the one we want to measure and which is found in the test
sample. The enzyme must be present in sufficient quantity so that it is not depleted
during the measurement.
The example of the determination of blood glucose by enzymatic method can be
developed by:
Hexokinase
Glucose oxidase
These are methods that make use of coupled reactions, involving a primary reaction
(transformation of the analyte of interest) and a second reaction called indicator
(formation of a compound with a measurable signal, colour, absorbance,
fluorescence, etc.).
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Principles of enzyme assays
• Hexokinase method for gycemia:
HK
Glucose + ATP → Glucose-6P +ADP
G6PDH
G6P + NAD+ → 6-Phosphogluconate + NADPH + H+
CLINICAL SIGNIFICANCE
Glucose is a major source of energy for most cells of the body; insulin
facilitates glucose entry into the cells.
Diabetes is a disease manifested by hyperglycemia; patients with
diabetes demonstrate an inability to produce insulin.
Clinical diagnosis should not be made on a single test result; it should
integrate clinical and other laboratory data.
The reagent contains HK and GPDH. The glucose present in the sample triggers the
reaction, measured proportionally by quantifying the NADH production in the second
reaction
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Principles of enzyme assays
• Method of Glucose Oxidase (GOx):
• Based on coupled reactions (GOx and Peroxidase) and
coloured product.
GOx
β-D-Glucose + H2O + O2 D-Gluconate + H2O2
H2O2
4-Aminoantipyrine + Phenol Quinoneimine + H2O
POD
Absorbance 500 nm
It employs two coupled enzymatic reactions. In the first, glucose is oxidized by action
of Glucose Oxidase (GOx) and H2O2 is generated. In the second, a peroxidase
(POD) decomposes H2O2, causing the oxidation of a chromogen to its colored form.
This chromogen is colorless in its reduced form and colored in its oxidized form. The
generation of the product is determined by a spectrophotometer (500nm).
The reagent kit contains the enzymes GOx and POD and the substrates necessary
for the reaction to take place. All of them are in defined and studied concentrations so
that the system works under optimal conditions. The reagent is added to the test
serum containing the glucose and the reaction is triggered. The color formed is then
measured with a spectrophotometer (500 nm). The technique is calibrated with
solutions of known glucose concentration with which the problem is compared.
At the present time, in the laboratories there are instruments destined to the
measurement of the enzymatic activities and that are able to store the factors
corresponding to each technique that they can carry out, they measure the increase
of absorbance and multiply this one by the factor informing of the activity of the
enzyme in study. If any failure has occurred during the reaction, this is also indicated.
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Principles of enzyme assays
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Technical aspects of enzyme-based
assays
• Regarding samples:
• Prevent protein denaturalization or damage
• Avoid freezing/thawing cycles but keep sample cool.
• Avoid the use of detergents in the assay tubes/materials
• Avoid hemolyzed samples
• Released enzymes from broken erythrocytes can change results.
• Fast separation of the clot
• Prevent the presence of enzymes from trombocytes
• Blood collection avoiding venous stasis (prolonged holding off of the
vein)
• It results in hypoxia and subsequent increase in the permeability of blood cells
• Avoid determination in aged samples
• Damage in certain enzymes (Creatin Kinase and Acid Phosphatase)
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Technical aspects of enzyme-based
assays
• Reagents and instruments
• Exhaustive control of expiration and opening dates and general state
• Keep storage and utilization conditions according to manufacturer specifications
(temperature, humidity, light, buffer reconstitution)
• Stick to the appropiate dilutions when required (linearity)
• In-time maintenance of instruments, manual or automated
• Maintenance and calibration regularly of micropipetes
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Frequent determinations and their role in
clinical analysis
• Phosphatases (hydrolases-esterases)
• Alkaline (pH 9) and acid(pH 5)
• Present in several tissues in different isoforms
• Alkaline: sign of liver damage/obstruction
• Acid: prostatic tumor marker
Frequent determinations
In the serum of patients, we will find, on the one hand, the so-called plasma-specific
enzymes, and on the other hand we will also detect physiological enzyme activities.
Plasma-specific enzymes carry out their function in the plasma. They are released
into the plasma by organs such as the liver (coagulation enzymes such as
prothrombin). If the organ responsible for their secretion is damaged, an alteration in
their levels would be detected.
Physiological enzyme activities have their origin in the cell renewal processes that
constantly take place in the organism, in muscular activity and in the passage of
enzymes from the secretory organs into the bloodstream. When there is a lesion in
the membrane of the cells, their contents, including intracellular enzymes, are
dumped into the interstitial space with a consequent increase in enzyme activity in the
serum.
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damage occurs and the detection of this increase in activity will be greater or lesser.
Thus we can deduce that knowledge of the cellular origin of enzymes, their
distribution and diffusion through the blood, lymph and interstitial tissue, as well as
their elimination pathway, allows us to obtain data of great interest in the diagnosis of
pathological phenomena.
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Frequent determinations and their role in
clinical analysis
Creatine kinase Creatine kinase MB
There are two kits for determining CK, both based on the same reactions. The
difference lies in the reagents:
The kit for CK determines the total activity, while the MB kit employs immuno-
inhibition for the suppression of the activity of the M sub unit, allowing the detection
of the activity corresponding to the B subunit.
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may be due to a lesion of the cardiac muscle or of another type of muscle. CK-MB
only appears in the blood in significant quantities when there is cardiac
damage.
If the CK value is elevated and the ratio of CK-MB to total CK (relative index) is
greater than 2.5 - 3, cardiac injury is likely. Elevated CK with a low relative index
suggests skeletal muscle injury. The percentage of CK-MB activity above 4%
should be considered suspicious and above 10% related to acute myocardial
infarction.
Any type of cardiac muscle injury can increase CK and CK-MB levels, such as
trauma, surgery, inflammation and ischemia. Strenuous physical exercise can also
increase CK and CK-MB, although in this case the relative index will be lower.
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Frequent determinations and their role in
clinical analysis
• Transaminases (transferases) of an α-amino to an α-ceto-
acid
AST (aspartate aminotransferase) is an enzyme found mainly in the liver, but also in
the muscles. When the liver is damaged, it releases AST into the bloodstream. The
AST blood test measures the amount of AST in the blood. It allows the doctor to
diagnose liver (hepatic) damage or disease.
ALT (alanine aminotransferase) is an enzyme found primarily in the liver. When liver
cells are damaged, they release this enzyme into the bloodstream. The ALT test
measures the level of ALT in the blood. Elevated levels of ALT in the blood can detect
a liver problem before there are signs of liver disease such as jaundice, which
causes the skin and eyes to turn yellow. The ALT blood test allows early detection of
liver disease.
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Frequent determinations and their role in
clinical analysis
• Transaminases (transferases) of an α-
amino to an α-cetoácido
• Gamma-glutamyl transferase (GT) of a γ-
Kinetic monitoring at 405nm
glutamyl to another peptide or aminoacid
• It is a microsomal enzyme, but levels are
frequently elevated in patients with liver
disease characterized by stasis of bile flow
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Determinaciones frecuentes y su
relevancia clínica
• Amylase α-Amylase
• Carbohydrate degradation 10 2-chloro-p-nitrophenyl-α-D-maltotrioside+ H2O
pH 6.0
enzyme
9 2-chloro-p-nitrophenol + glucose + α-D-maltoside
• Two isoforms,-P (pancreatic) and
S (salivary glands)
• Diagnosis of pancreatic illness
(acute pancreatitis) in
combination with lipase (blood).
• Samples are serum, plasma
(heparinized) and urine
Activity: how to detect urea in blood samples and what is its clinical importance?
Most patients with acute pancreatitis have elevations in serum levels of amylase or
lipase within a few hours of the onset of symptoms. Lipase is generally preferred over
amylase as a diagnostic test because of its superior specificity
Amylase and lipase levels also can be elevated in a variety of other conditions that
may mimic acute pancreatitis, including intestinal ischemia and infarction, bowel
obstruction, cholecystitis, and choledocholithiasis. In addition, amylase levels may be
elevated from ectopic pregnancy, acute salpingitis, and a variety of extra-abdominal
conditions such as parotitis, lung cancer, and head trauma.
In this direct assay the α-amylase catalyzes the hydrolysis of the substrate 2-chloro-p-
nitrophenyl-α-D-maltotrioside (CNP-G3) at pH 6.0 to 2-chloro-p-nitrophenol (CNP)
and free glycosides. The reaction is controlled kinetically at 405 nm from the rate of
color formation of the produced CNP proportional to the α-amylase activity in the
sample.
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Enzyme Type of analysis Sample Reaction Application
Resumen de determinaciones
Acid Phosphatase
Spectrophotometric endpoint, or
kinetic. (colorimetric 405 nm)
Clear, free of hemolysis serum,
separated from the clot,
immediately.
Prostatic cancer diagos
enzimáticas
Alanine
aminotransferase
Spectrophotometric, kinetic.
Monitoring NADH at 430 nm.
Serum, EDTA or heparinized
plasma free of hemolysis
Liver damage
Spectrophotometric endpoint, or
Serum, EDTA or heparinized Liver and muscle, infarction.
Aldolase kinetic. Monitoring NADH at 430 plasma Tracking of muscular dystrophy
nm.
*2-cloro-p-nitrofenil-α-D-maltotriósido/**2-cloro-p-nitrofenol
Liver damage, myocardial
Aspartate Spectrophotometric , kinetic. Serum, EDTA or heparinized infarction, muscular dystrophy,
aminotransferase Monitoring NADH at 430 nm. plasma free of hemolysis pancreatitis
Indicator of myocardial
Free of hemolysis serum infarction (8-12h to 4-5days),
separated from the clot, pulmonary embolism, kidney
Lactate Spectrophotometric. Monitoring
immediately. Heparin and citrate disease(tubular necrosis or
dehydrogenase NADH at 430 nm. produce a false LDH activity pyelonephritis), abdominal and
increase lung cancer. Muscular
dystrophy, anaemia
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