Download enzymes

2014-10-06
ENZYMES
as biological catalysts in
chemical reactions
Outline
• Composition, structure and properties of enzyme
• How Enzymes work
• Enzyme activity
• Factors affecting enzyme activity
• Regulation of enzyme activities
• Enzymes in clinical diagnosis
•Coenzymes and cofactors
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Three criteria must be met for a chemical reaction to
occur:
• The reactants, called substrates must collide
• The molecular collision must occur with the
correct orientation
• The reactant must have sufficient energy. This
energy is called the activation energy
Enzymes are Biological Catalysts
Enzymes are proteins that:
• Increase the rate of reaction by
lowering the energy of activation.
• Catalyze nearly all the chemical
reactions taking place in the cells
of the body.
• Have unique three-dimensional
shapes that fit the shapes of
reactants.
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Enzyme Catalyzed Reaction
• The proper fit of a substrate (S) in an active site forms
an enzyme-substrate (ES) complex.
E+S
ES
• Within the ES complex, the reaction occurs to convert
substrate to product (P).
ES
E+P
• The products, which are no longer attracted to the
active site, are released.
• Overall, substrate is convert to product.
E+S
ES
E+P
Enzyme-Substrate Complex
• Substrates bind to a depression on the surface of enzymes
known as the active site, to form an enzyme-substrate
complex.
• The active site undergoes a slight conformation change to
better accommodate the substrate (induced fit).
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Active Site
The active site:
• Is a region within an
enzyme that fits the shape of
molecules called substrates.
• Contains amino acid R
groups that align and bind
the substrate.
• Releases products when the
reaction is complete.
Some key features of the active site
• The active site takes up a relatively small part of the
total volume of an enzyme
• The active site is a three-dimensial entity
• Substrates are bound to enzymes by multiple weak
attraction
• Active sites are clefts or crevices
• The specificity of binding depends on the precisely
defined arrangement of atoms in an active site
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The substrate binding site
• The substrate binding site is a particular arrangement of
chemical groups on the enzyme surface that is specially
formulated to bind a specific substrate
• The substrate binding site may have been integrated within it
the active site or in some cases the active site may not be within
the substrate binding site
Binding uses multiple weak interactions
1.
2.
3.
4.
Hydrogen bonds
Salt links
van der Waals interactions
Hydrophobic effect
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The allosteric site
• The allosteric site is not at the active site or substrate binding
site, but is somewhere else on the molecule
• The allosteric site is the site where small molecules bind and
affect a change in the active site or the substrate binding site
• The binding of this specific molecule causes a change in the
conformation of enzyme what cause the active site to become
either more or less active
• It may cause the binding site to have a greater affinity for
substrate or have less affinity for substrate
Enzyme Specificity
Enzymes may recognize and catalyze:
• A single substrate.
• A group of similar substrates.
• A particular type of bond.
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•Trypsin, chymotrypsin, and elastase all carry out the same
reaction — the cleavage of a peptide chain, but they display very
different specificities.
•Trypsin cleaves peptides on the carbonyl side of the basic amino
acids, arginine or lysine.
•Chymotrypsin prefers to cleave on the carbonyl side of aromatic
residues, such as phenylalanine and tyrosine.
• Elastase is not as specific as the other two; it mainly cleaves
peptides on the carbonyl side of small, neutral residues.
Classification of Enzymes
•
Enzymes are classified according to the reaction they
catalyze.
Class
Reactions catalyzed
1. Oxidoreductases
Oxidation-reduction
2. Transferases
Transfer groups of atoms
3. Hydrolases
Hydrolysis
4. Lyases
Add atoms/remove atoms
to/from a double bond
5. Isomerases
Rearrange atoms
6. Ligases
Use ATP to combine molecules
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The following classification names apply:
•
•
•
•
• Oxidoreductases
These are enzymes that catalyze oxidations or
reductions.
Enzymes such as dehydrogenases, oxidases, and
peroxidases.
• Transferases
These enzymes catalyze the transfer of a group from
one molecule to another.
Examples such as phosphatases, transaminases, and
transmethylases.
Classification of Enzymes: Oxidoreductases and
Transferases
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•
•
•
•
• Hydrolases
These enzymes catalyze hydrolysis reactions.
Examples are the digestive enzymes such as sucrase,
amylase, maltase, and lactase.
• Lyases
These enzymes catalyze the removal of groups in
non-aqueous media.
An example would be the decarboxylases.
Classification: Hydrolases and Lyases
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• Isomerases
• Enzymes that catalyze the isomerization of
molecules.
• Examples are racemases, and cis-trans isomerases.
• Ligases
• These are also called synthetases which are enzymes
that catalyze condensation reactions where smaller
molecules are connected with the resulting removal of
a water molecule.
• This is accompanied with the formation of a high
energy phosphate link that stores energy.
• An example would be the amino acid RNA ligases.
Classification: Isomerases and Ligases
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Two Models for Enzyme-Substrate Interaction
Lock-and-Key Model
In the lock-and-key model of enzyme action:
• The active site has a rigid shape.
• Only substrates with the matching shape can fit.
• The substrate is a key that fits the lock of the active
site.
Induced-fit Model
In the induced-fit model of enzyme action:
• The active site is flexible, not rigid.
• The shapes of the enzyme, active site, and substrate
adjust to maximum the fit, which improves catalysis.
• There is a greater range of substrate specificity.
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Induced Conformational Change in
Hexokinase
The Michaelis-Menten equation is the basic equation of
enzyme kinetics
• For many enzymes, the rate
of catalysis, V varies with
substrate concentration, [S],
in a following manner
Vmax [S ]
V=
(K m + [S ])
Km describes the substrate concentration at which the reaction velocity is halfmaximal
Vmax occurs at high substrate concentrations when the enzyme is sturated, that
is, when it is entirely in the ES form
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It is convenient to transform the M-M equation into
one that gives a straight line plot
Vmax [S ]
V=
(K m + [S ])
• Simplifying the above equation gives the
Lineweaver-Burk relationship
• Taking the reciprocal of
both sides of the
equation yields
Plotting the reciprocals of the same data points yields a "double-reciprocal"
or Lineweaver-Burk plot.
This provides a more precise way to determine Vmax and Km.
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Plots of 1/v versus 1/[S] yield straight lines having a slope of
Km/Vmax and an intercept on the ordinate at 1/Vmax
Factors affecting enzyme activity
•
•
•
•
•
•
Concentration of substrate
Concentration of enzyme
Temperature
pH
Activators
Inhibitors
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Substrate Concentration
• The rate of reaction
increases as substrate
concentration increases (at
constant
enzyme
concentration).
• Maximum activity occurs
when the enzyme is
saturated.
pH and Enzyme Action
Enzymes:
• Are most active at optimum
pH.
• Contain R groups of amino
acids with proper charges at
optimum pH.
• Lose activity in low or high
pH as tertiary structure is
disrupted.
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Optimum pH Values
• Most enzymes of the body have an optimum pH of
about 7.4.
• In certain organs, enzymes operate at lower and higher
optimum pH values.
Temperature and Enzyme Action
Enzymes:
• Are most active at an
optimum
temperature
(usually 37°C in humans).
• Show little activity at low
temperatures.
• Lose
activity
at
high
temperatures as denaturation
occurs.
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Temperature and Enzyme Action
• As the temperature rises, reacting molecules have
more and more kinetic energy.
• This increases the chances of a successful collision
and so the rate increases.
• Above this temperature the enzyme structure begins
to break down (denature) since at higher temperatures
intra- and intermolecular bonds are broken as the
enzyme molecules gain even more kinetic energy.
Enzyme Concentration
• The rate of reaction
increases as enzyme
concentration increases
(at constant substrate
concentration).
• At
higher
enzyme
concentrations,
more
substrate binds with
enzyme.
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TERMINOLOGY
• Enzyme activity is usually expressed in units of µmol of
substrate converted to product per minute under specific assay
conditions
• One standard unit of enzyme activity (U) is an amount of
activity that catalyzes the transformation of 1 µmol/min
• The specific activity is defined as the number of enzyme units
per mg of protein (U/mg of protein)
• The catalytic constant Kcat or turnover number is equal to the
units of enzyme activity per mol of enzyme
• Katal (kat) denotes the conversion of 1 mol substrate per
second
• The maximum velocity Vmax is the velocity obtained under
condition of substrate saturation of the enzyme under a given
set of condition of pH, temperature,
Enzyme Inhibition
• Inhibitors are molecules that reduce the rate of
enzymatic reactions
• The are usually specific and they work at low
concentrations
• They block the enzyme but they do not usually
destroy it
• Many drugs and poisons are inhibitors of enzymes in
the nervous system
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Types of inhibitors
• Enzyme Inhibitors can be divided into two
major types:
Reversible inhibitors and irreversible
inhibitors
Reversible Versus Irreversible Inhibition
• Reversible inhibitors interact with the enzyme
through noncovalent association/dissociation
reactions.
• In contrast, irreversible inhibitors usually cause
stable, covalent alterations in the enzyme.
• That is, the consequence of irreversible inhibition is a
decrease in the concentration of active enzyme.
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The effect of enzyme inhibition
1. Competitive:
•
Inhibitor competes with the
substrate molecules for the
E+I
active site of enzyme
Reversible
•
The inhibitor’s action is
proportional to its concentration reaction
•
Inhibitor resembles the
substrate’s structure closely
Has its effect reversed by
increasing substrate
concentration.
EI
Enzyme inhibitor
complex
Double Reciprocal Plots Facilitate evaluation of
inhibitors
Figure represents a typcal case of competitive inhibition shown
graphically in the form of Lineweaver-Burk plot
Competitive inhibitor
increases Km but
not changes Vmax
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Malonate and Succinate Dehydrogenase
Succinate
Succinate
dehydrogenase
Fumarate + 2H++ 2e-
Malonate:
• Is a competitive inhibitor
of succinate
dehydrogenase.
• Has a structure that is
similar to succinate.
• Inhibition is reversed by
adding succinate.
Ethanol as inhibitor in methanol and ethylene glycol
poisonings
Methanol is relatively non-toxic; however, it is metabolized to highly toxic
compounds that are responsible for the acidosis and blindness characteristic
of methanol poisoning.
Ethanol is a competing substrate and so it blocks the oxidation of methanol to
aldehyde products
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Noncompetitive Inhibition
A noncompetitive inhibitor:
• Has a structure different than the substrate.
• Distorts the shape of the enzyme, which alters the
shape of the active site.
• Prevents the binding of the substrate.
• Cannot have its effect reversed by adding more
substrate.
Noncompetitive Inhibition
Lineweaver-Burk plot for reversible noncompetitive inhibition
Noncompetitive inhibitor
decreases Vmax but not changes Km
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Uncompetitive inhibition. In uncompetitive inhibition
inhibitor binds only to enzyme-substrate complex.
Uncompetitive inhibition decreases the maximum velocity
(Vmax) as well as the KM
Irreversible Inhibition
• In irreversible inhibition, a substance destroys enzyme
activity by bonding with side-chain groups in(R) the
active site .
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Regulation of enzyme activity
Regulation of enzyme activity is achieved in a variety
of ways:
• ranging from controls over the amount of enzyme
protein produced by the cell
• to more rapid, reversible interactions of the enzyme
with metabolic inhibitors and activators.
Zymogens
• Zymogen: Inactive enzyme precursor, cleavage of one or more
covalent bonds transforms it into active enzyme
• Chymotrypsinogen
– synthesized and stored in the pancreas
– a single polypeptide chain of 245 amino acid residues cross
linked by 5 disulfite bonds
– when secreted into the small intestine, the digestive enzyme
trypsin cleaves a 15 unit polypeptide from the N-terminal end to
give α chymotrypsin (active form)
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Digestive Enzymes
Digestive enzymes are:
• Produced as zymogens in one organ and transported to
another such as the pancreas when needed.
• Activated by removing small peptide sections.
Allosteric Enzymes
• Allosteric: Greek allo + steric, other shape
• Allosteric enzyme: an oligomer whose biological
activity is affected by other substances binding to it
– these substances change the enzyme’s activity by
altering the conformation(s) of its 4°structure
• Allosteric effector: a substance that modifies the
behavior of an allosteric enzyme; may be an
– allosteric inhibitor
– allosteric activator
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Allosteric Enzymes
• An allosteric enzyme is an enzyme in a reaction
sequence that binds a regulator substance.
• A positive regulator enhances the binding of
substrate and accelerates the rate of reaction.
• A negative regulator prevents the binding of the
substrate to the active site and slows down the rate
of reaction.
A change in shape
• When the inhibitor is present it fits into its site
and there is a conformational change in the
enzyme molecule
• The enzyme’s molecular shape changes
• The active site of the substrate changes
• The substrate cannot bind with the active site
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Switching off
• These enzymes
have two receptor
sites
• One site fits the
substrate like other
enzymes
• The other site fits
an inhibitor
molecule
Substrate
cannot fit into
the active site
Inhibitor
molecule
Inhibitor fits into
allosteric site
Phosphorylation /dephosphorylation
•most common covalent modification
• involve protein kinases/phosphatase
•PDK (pyruvate dehydrogenase) inactivated by
phosphorylation
•Amino acids with –OH groups are targets for
phosphorylation
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Feedback Control
In feedback control:
• A product acts as a negative regulator.
• An end product binds with the first enzyme (E1) in a
sequence, when sufficient product is present.
Diagnostic Enzymes
• The levels of diagnostic enzymes determine the
amount of damage in tissues.
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Isoenzymes
• Isoenzymes are enzymes that differ in amino acid
sequence but catalyze the same chemical reaction.
• These enzymes usually display different kinetic
parameters (e.g. different KM values), or different
regulatory properties
• In clinical medicine, isoenzyme has a more restricted
meaning, namely, the physically distinct and
separable forms of a given enzyme present in
different cell types or subcellular compartments of a
human being.
Lactate dehydrogenase (LDH)
• Lactate dehydrogenase isoenzymes differ at the level
of quaternary structure
• The oligometric LDH molecule consists of
4 protomers of 2 types, H and M.
• Only the tetrameric molecule possesses catalytic
activity.
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Isoenzymes
• Isoenzymes
catalyze
the same reaction in
different tissues in the
body.
• Lactate dehydrogenase,
which converts lactate to
pyruvate, (LDH) consists
of five isoenzymes.
Enzyme Cofactors
•
•
•
A simple enzyme is an active enzyme that consists only of protein.
Many enzymes are active only when they combine with cofactors such
as metal ions or small molecules.
A coenzyme is a cofactor that is a small organic molecule such as a
vitamin.
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Metal ion catalysts
One-third of all known enzymes needs metal ions to work!!
1. Metalloenzymes: contain tightly bound metal ions: I.e. Fe++,
Fe+++, Cu++, Zn++, Mn++, or Co++.
2. Metal-activated enzymes- loosely bind ions Na+, K+, Mg++, or
Ca++.
They participate in one of three ways:
They bind substrates to orient then for catalysis
Through redox reactions gain or loss of electrons.
Electrostatic stabilization or negative charge shielding
Vitamin Coenzymes
Vitamin
Thiamine (B1)
Riboflavin (B2)
Coenzyme Made
Thiamine
Pyrophosphate
FAD, FMN
folic acid
tetrahydrofolic acid
biotin
biocytin
Function
Decarboxlation
Electron Transfer
amino acid metabolism
CO2 fixation
Pantothenic Acid
Coenzyme A
acyl group carrier
Ascorbic Acid (C)
Vitamin C
Collagen synthesis
Healing
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