Download BioN04 Enzymes 2015 v2

Enzymes
Biochemistry for Nursing
Summer semester, 2015
Dr. Mamoun Ahram
What are enzymes?
• Enzymes are specialized proteins that are able to
conduct chemical reactions under biological
conditions.
• Most enzymes have very specific functions, and
convert specific substrates to the corresponding
products.
Enzymes are catalysts
• Enzymes are catalysts.
– They increase the rate of a reaction.
– Enzymes accelerate reactions by factors of as much as
a million or more.
• They are used in small amounts relative to the
reactants.
• They are not consumed in the reaction.
• Enzymes do not change an energetically unfavorable
into a favorable one.
How to express an enzymatic reaction?
• Reactants are known as substrates.
• We can express an enzymatic reaction like this:
E + S ES  EP  E + P
where E is the free enzyme; S is the free substrate, ES is the
enzyme-substrate complex; P is the product of the reaction; and
EP is the enzyme-product complex before the product is released
• To simplify the reaction, the reaction is expressed as
E + S ES  E + P
Turnover number
• A simple reaction of the hydration of carbon dioxide is catalyzed by
an enzyme called carbonic anhydrase.
• Each enzyme molecule can hydrate 106 molecules of CO2 per
second.
• The catalyzed reaction is 107 times as fast as the uncatalyzed one.
• The catalytic activity of an enzyme is measured by its turnover
number, the maximum number of substrate molecules converted
by one molecule of enzyme per unit time.
General properties of enzymes
• The function of nearly all proteins depends on their
ability to bind other molecules (ligands).
• Two properties of a protein in regards to its
interaction with ligands:
– affinity: the strength of binding between a protein and
other molecules
– specificity: the ability of a protein to bind one
molecule in preference to other molecules
Examples of very specific enzymes
Proteases
Some enzymes are less specific
Carbxypeptidase
Active sites of enzymes
• Each enzyme has a specific three-dimensional shape
that includes a region where the biochemical
reaction takes place, called the active site
• The active site contains a specialized amino acid
sequence that facilitates the reaction.
Features of active site 1
• Binding is specific due to the precise interaction of
the substrate with the enzyme.
• Binding occurs at least at three
points.
• This illustrates the importance of
chirality.
• If a substrate is chiral, an
enzyme usually catalyzes the
reaction of only one of the pair
of enantiomers because only
one fits the active site in such a
way that the reaction can
occur.
Features of active site 2
• The active site is small.
• The “extra” amino acids:
– create the threedimensional structure of
active site,
– form regulatory sites that
interact with other
proteins or small
molecules and control the
activity of the enzyme.
Features of active site 3
• Active sites are structures that look like canals that
bind substrate molecules.
Features of active
site 4
• Substrates are bound to
enzymes by multiple weak
attractions including :
–
–
–
–
electrostatic interactions,
hydrogen bonds,
van der Waals forces, and
hydrophobic interactions.
Naming of enzymes
• In general, enzymes end with the suffix (-ase)
• Most other enzymes are named for their substrates and
for the type of reactions they catalyze, with the suffix
“ase” added
– An ATPase is an enzyme that breaks down ATP
– ATP synthase is an enzyme that synthesizes ATP
• Some enzymes have common names that provide little
information about the reactions that they catalyze
• Examples include the proteolytic enzyme trypsin
Classification of enzymes
• Enzymes were classified into six major groups
–
–
–
–
–
–
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
Oxidoreductases
•
•
These enzymes catalyze oxidation and reduction
reactions involving the transfer of hydrogen atoms
or electrons
This group can be further divided into 4 main
classes:
–
–
–
–
Dehydrogenases
Oxidases
Peroxidases
Oxygenases
Dehydrogenases
•
•
•
Dehydrogenases catalyze Removal of 2 hydrogens to
form a double bond .
Usually, hydrogen is transferred from the substrate to a
molecule known as nicotinamide adenine dinucleotide
(NAD+)
Lactate dehydrogenase catalyzes this reaction
Alcohol dehydrogenase
• Another example is alcohol dehydrogenase
Oxidases
•
•
Oxidases catalyze hydrogen transfer from the
substrate to molecular oxygen producing hydrogen
peroxide (H2O2) as a one product (or addition of O2
to a substrate)
Glucose oxidase catalyzes this reaction:
-D-glucose + O2  gluconolactone + H2O2
Peroxidases
•
•
Peroxidases catalyze
oxidation of a substrate
by hydrogen peroxide.
Example: the oxidation
of two molecules of
glutathione (GSH) in
the presence of
hydrogen peroxide:
Oxygenases
•
•
•
Oxygenases catalyze substrate oxidation by oxygen
The reduced product of the reaction in this case is
water and not hydrogen peroxide
An example of this is the oxidation of lactate to acetate
catalyzed by lactate-2-monooxygenase
CH3-CH(OH)-COOH + O2  CH3COOH + CO2 + H2O
Transferases
• These enzymes transfer a functional group (C, N, P or S)
from one substrate to an acceptor molecule
• Types: transaminases and kinases
Transaminases
• A transaminase transfers an amino functional group
from one amino acid to a keto acid, converting the
amino acid to a keto acid and the keto acid to an
amino acid
• This allows for the interconversion of certain amino
acids
Kinases transfer of a phosphoryl
group between substrates
Hydrolases
• These enzymes catalyze cleavage reactions while
using water
• Peptidases, esterases, lipases, glycosidases,
phosphatases are all examples of hydrolases named
depending on the type of bond cleaved
Proteases
• A class of hydrolytic enzymes is proteases
• These enzymes catalyze proteolysis, the hydrolysis of
a peptide bond within proteins
• Proteolytic enzymes differ in their degree of
substrate specificity
Exmaples
• Trypsin, a digestive enzyme, is quite specific and
catalyzes the splitting of peptide bonds only on the
carboxyl side of lysine and arginine residues
• Thrombin, an enzyme that participates in blood
clotting, catalyzes the hydrolysis of Arg-Gly bonds in
particular peptide sequences only
Lyases
• These enzymes remove groups from their substrates
resulting in the formation or removal of double
bonds between C-C, C-O and C-N by a means other
than hydrolysis
Types of lyases
• Dehydrases: Removal of H2O
from substrate to give double
bond (example: enolase)
• Decarboxylases: Replacement of
a carboxyl group by a hydrogen
• (example: pyruvate
decarboxylase)
• Synthases: Addition of a small
molecule to a double bond
(example: citrate synthase)
Isomerases
• These enzymes catalyze intramolecular rearrangements
Ligases
• Ligases join C-C, C-O, C-N, C-S and C-halogen bonds
• The reaction is usually accompanied by the
consumption of a high energy compound such as ATP
and other nucleoside triphosphates
• Example: pyruvate carboxylase, which catalyzes:
Pyruvate + HCO3- + ATP  Oxaloacetate + ADP + Pi
HOW DO SUBSTRATES FIT INTO THE
ACTIVE SITE OF ENZYMES?
Lock-and-key model
• The first is known as lock-and-key model where the
substrate fits directly into the active site.
Lock-and-key model
Induced fit m
Induced fit model
modelas
• TheLock-and-key
other model known
induced fit model states
that enzymes are flexible
and that the shapes of the
active sites can be changed
by the binding of substrate.
Induced fit model
Activation energy
• Conversion of a substrate to a product requires an
energy called activation energy.
• Enzymes accelerate reactions by lowering this energy.
Binding of substrate
to an enzyme makes
it less stable
ENZYME REGULATION
Mechanisms of regulation
• Non-specific inhibition (pH and temperature)
• Regulation of substrate and enzyme amount
• Location (Compartmentalization and complexing of
enzymes)
• Expression of enzyme type (isoenzymes)
• Regulation of enzymatic activity
•
•
•
•
Inhibitors
Allostery
Reversible covalent modification
Irreversible covalent modification
Effect of substrate Concentration
• The rate increases with more
substrate added to the
reaction because more
enzyme molecules are bound
by the substrate.
– If the substrate
concentration doubles, the
rate of the reactions
doubles.
• But: the increase in the rate
begins to level off because
the active sites are occupied
(they become saturated).
The rate of the reaction is
determined by the efficiency
of the enzyme, the pH, and
the temperature
Saturation
Effect of Enzyme Concentration
• If the enzyme
concentration doubles,
the rate doubles; if the
enzyme concentration
• triples, the rate triples;
and so on.
Regulation of enzyme amount
• There are basically two mechanisms:
– controlling rate of enzyme synthesis at the gene level
– controlling rate of enzyme degradation by proteases
Temperature
• Usually, the reaction rate
increases
• But, at high temperatures,
the activity decreases
because the protein part of
the enzyme begins to
denature
• Enzymes have an optimal
temperature
– For humans, it is 37°C
– For thermophilic bacteria, it
can be 65°C
Hypothermia
• A severe drop in body temperature creates the potentially
fatal condition of hypothermia, which is accompanied by a
slowdown in metabolic reactions.
• This effect is used to advantage by cooling the body during
cardiac surgery.
• Upon gentle warming, enzymatic reaction rates return to
normal because cooling does not denature proteins.
pH
• pH can alter binding of substrate to enzyme by
altering the protonation state of the substrate and/or
altering the conformation of the enzyme
• The effect of pH is enzyme-dependent
Enzymes can be placed in small
cellular compartments
• For example, lysosomes contain many of the
proteolytic enzymes responsible for protein
degradation
• Another example, synthesis of fatty acids are located
in cytosol, whereas enzymes responsible for
oxidation (break-sown) of fatty acids are located in
the mitochondria
Enzymes can be made of multiple
enzymes
• The enzymes involved in a reaction sequence form a
multienzyme complex
– This allows the product of enzyme A to be passed
directly to enzyme B
• Example: Pyruvate dehydrogenase
Pyruvate + CoA + NAD+ Acetyl CoA + CO2 + NADH
Allostery
• Allosteric regulation: binding of one molecule to one
site changes binding of another molecule on a
different site
• Allosteric enzymes tend to be a multi-subunit
• Allosteric enzymes contain an active site and a
regulatory site
• The binding of regulatory molecules causes
conformational changes in the active site
Allosteric control
Negative
allosteric
regulation
Positive
allosteric
regulation
Enzyme inhibitors
• The activity of many enzymes can be inhibited by the
binding of specific small molecules.
• Enzyme inhibition can be either reversible or
irreversible.
• Physiological inhibitors are reversible.
Irreversible inhibitors
• An irreversible inhibitor tightly binds to the enzyme
preventing the substrate from binding.
• The inhibitor forms a bond that is not easily broken
with a group in an active site.
• Examples:
– Heavy metals like mercury bind to –SH groups of
cysteine
– Sarin (nerve gas) binds to serine in the active site of
acetylcholinesterase inhibiting it (no ore nerve
impulses, paralysis)
Reversible inhibitors
• Reversible inhibition is characterized by a rapid
dissociation of the enzyme-inhibitor complex
• Usually these inhibitors bind to enzymes by noncovalent forces
Types of reversible inhibitors
Competitive
• The inhibitor competes with
the substrate for the active
site
Noncompetitive
• the inhibitor binds at a site
other than the catalytic site
Reversible covalent modification
• A small group is added to the enzyme affecting its
function.
• Advantage: fast and transient regulation of enzyme
activity
The addition (phosphorylation)
or removal (dephosphorylation)
of a phosphate group by
enzymes may activate or
inactivate proteins like other
enzymes
Irreversible covalent modification
(proteolytic activation)
• Many enzymes are synthesized as inactive precursors called
zymogens or proenzymes
• When a substrate is present, part of the enzyme is removed to
make it an active enzyme
• However, it is an irreversible process, so once the pro region is
removed, it cannot go back
Examples of zymogens
(digestive enzymes)
How does it work?
• Three of the enzymes that digest proteins in the small
intestine are produced in the pancreas as the zymogens
trypsinogen, chymotrypsinogen, and proelastase.
• These enzymes are inactive when they are synthesized so that
they do not digest the pancreas.
• Each zymogen has a polypeptide segment at one end that is
not present in the active enzymes.
• The extra segments are snipped off to produce trypsin,
chymotrypsin, and elastase, the active enzymes, when the
zymogens reach the small intestine, where protein digestion
occurs.
Acute pancreatitis
• One danger of traumatic injury to the pancreas or
the duct that leads to the small intestine is
premature activation of these zymogens, resulting in
acute pancreatitis, a painful and potentially fatal
condition in which the activated enzymes attack the
pancreas.
Isoenzymes (isozymes)
• These are enzymes that
– Are produced by different genes
– Can act on the same substrate(s) producing the same
product(s)
– Have different tissue distribution
– Have different mechanisms of regulation
– May have different catalytic activity
LDH
• Lactate dehydrogenase (LDH) is a tetrameric enzyme
composed of two protein subunits; the subunits are known as
H (for heart) and M (for skeletal muscle)
• These subunits combine in various combinations leading to 5
distinct isozymes
• These subunits combine in various combinations leading to 5
distinct isozymes (LDH1-5) with different combinations of the
M and H subunits
• The all H isozyme is characteristic of that from heart tissue,
and the all M isozyme is typically found in skeletal muscle and
liver
MODES OF REGULATION
Feedback inhibition
• A common type of control
occurs when an enzyme
present early in a
biochemical pathway is
inhibited by a late
product of pathway
• This is known as feedback
inhibition or negative
feedback regulation
Positive feedback regulation
• Enzymes can also be subject to positive feedback
regulation where a product stimulates the activity of
an enzyme
Feed-forward regulation
• A third mechanism is feed-forward regulation where
a substrate produced early in a pathway activates an
enzyme downstream of the same pathway
A committed step
• A committed step is an irreversible reaction that,
once occurs, leads to the formation of a final
substrate with no point of return
• For example, the committed step for making product
E is (B → C), not (A → B)
A
C
D
E
X
Y
Z
B
Rate-limiting reactions
• Some reactions are called rate-limiting since they
limit rate (speed) of reactions.
• Why do they limit reactions?
– They need lots of energy.
– They are highly regulated.
ENZYMES IN DISEASE DIAGNOSIS
Concept
• The measurement of the serum levels of numerous
enzymes has been shown to be of diagnostic
significance
• This is because the presence of these enzymes in the
serum indicates that tissue or cellular damage has
occurred resulting in the release of intracellular
components into the blood
Information from enzymes
measurements in serum
•
•
•
•
Presence of disease
Organs involved
Etiology /nature of disease: differential diagnosis
Extent of disease-more damaged cells-more leaked
enzymes in blood
• Time course of disease
Measurement of enzyme activity
• Enzyme activity is expressed in International unit (IU)
• It corresponds to the amount of enzymes that
catalyzes the conversion of one micromole (mol) of
substrate to product per minute
Enzymes used in the clinic
ENZYME
PRESENT IN
Aspartate Amino transferase (AST)
Serum glutamate-oxaloacetate
transaminase (SGOT)
Heart and Liver
Alanine Amino transferase (ALT)
Serum glutamate-pyruvate
transaminase (SGPT)
Heart and Liver
Alkaline Phosphatase (ALP)
Bone, intestine and other tissues
Acid Phosphatase (ACP)
Prostate
 glutamyl Transferase ( GT)
Liver
Creatine kinase (CK)
Muscle Including cardiac muscle
Lactate Dehydrogenase (LDH)
Heart, liver, muscle, RBC
 Amylase
Pancreas
AST and ALT
• The typical liver enzymes measured are AST and ALT
• ALT is particularly diagnostic of liver involvement as
this enzyme is found predominantly in hepatocytes.
• When assaying for both ALT and AST the ratio of the
level of these two enzymes can also be diagnostic.
• Normally in liver disease or damage that is not of
viral origin the ratio of ALT/AST is less than 1.
• However, with viral hepatitis the ALT/AST ratio will be
greater than 1.
ALKALINE PHOSPHATASE (ALP)
• Is a group of enzymes that have maximal activity at a
high pH 9.0-10.5
• High levels are seen is liver and bone, and useful to
assess hepatobiliary and bone diseases
• In liver, high levels of ALP is indicative of extrahepatic
obstruction
• In bones, the enzyme is increased in bone diseases like
rickets, osteomalacia, neoplastic diseases with bone
metastates and healing fractures
ACID PHOSPHATASE (ACP)
• Is a group of enzymes that have maximal activity at
pH 5.0-6.0
• It is present in prostate gland.
• The main source of ACP is prostate gland and so can
be used as a marker for prostate disease.
AMYLASE
• Is the digestive enzymes from the pancreas and
salivary glands to digest complex carbohydrates.
• Elevated in acute pancreatitis.
• It is used as a marker to detect acute pancreatitis
and appendicitis.
-Glutamyltransferase (-GT)
• Amino acid + Glutathione   -glutamyl amino acid +
Cysteinylglycine
• Found mainly in biliary ducts of the liver, kidney and pancreas.
• Enzyme activity is induced by a number of drugs and in
particular alcohol.
• -GT increased in liver diseases especially in obstructive
jaundice.
• -GT levels are used as a marker of alcohol induced liver
disease and in liver cirrhosis.
Myocardial infarction
CPK
• CPK is found primarily in heart and skeletal muscle as
well as the brain. Therefore, measurement of serum
CPK levels is a good diagnostic for injury to these
tissues
• Like LDH, there are tissue-specific isozymes of CPK:
– CPK3 (CPK-MM) is the predominant isozyme in muscle
– CPK2 (CPK-MB) accounts for about 35% of the CPK
activity in cardiac muscle, but less than 5% in skeletal
muscle.
– CPK1 (CPK-BB) is the characteristic isozyme in brain
and is in significant amounts in smooth muscle
CPK and myocardial infarction
• Since most of the released CPK after a myocardial
infarction is CPK-MB, an increased ratio of CPK-MB to
total CPK may help in diagnosis of an acute
infarction, but an increase of total CPK in itself may
not
LDH
• Lactate dehydrogenase exists in 5 types differentially
and specifically distributed in tissues
• The 5 isoforms of LDH: LDH1, 2, 3, 4, and 5
• LDH is especially diagnostic for myocardial infarction
(heart attacks) among other diseases like acute
hepatitis (LDH5 > LDH4).
LACTATE DEHYDROGENASE IN MI
LDH1 vs. LDH2
• A characteristic change
following a heart attack is
an elevated level of LDH1
above LDH2.
• A comparison of serum
levels of LDH-1/LDH-2 ratio
is diagnostic
• Normally, this ratio is less
than 1
• Following an acute
myocardial infarct, the LDH
ratio will be more than 1.