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Introduction
Enzymes are the catalysts of biological systems
Catalystis a substance that accelerate the rate of a chemical reaction without it being changed during
.The process
.Most enzymes are proteins that accelerate chemical reactions by factors of at least million
They catalyze hundreds of stepwise reactions by which nutrient molecules are degraded, chemical
Energy is conserved and transformed and biological macromolecules are made from simple
.Precursors
)2Enzymes are very specific to their substrates . This way the processes are very efficient and there is no loss
.of energy
DNA polymerase I synthesizes very accurately DNA strands . It can make only one mistake in a million
.Because it proofreads the product during synthesis and corrects its mistakes
)3The catalytic activities of many enzymes are regulated
A)Feedback inhibition : An enzyme that catalyzes the first step in a biosynthetic pathway is inhibited by the end
product. When the concentration of the end product is high enough it can bind directly to the enzyme and
inhibit its activity. When the level of the end products drops, it will be released and allow the enzyme to
.function
B)Regulatory proteins : Proteins can bind to enzymes and regulate their activities. The activities of many enzymes
+is regulated by a small protein Calmodulin. Calmodulin is a sensor of Ca2+ in the cell. The binding of Ca2
to multiple sites in calmodulin induces a conformational change that converts it to an active state. It can then
.bind many enzymes and modify their activities
Myosin light
Chain kinase
D) Proteolytic activation: Some enzymes are synthesized as inactive precursors and are activated in the appropriate
time and place by proteolytic cleavage. For example trypsinogen is synthesized in the pancreas and is activated
.by proteolytic cleavage in the small intestine to form an active trypsin. This mechanism is unidirectional
In happens also in blood clotting. The inactive precursor is called.Zymogene
)4Enzymes transform different forms of energy): *Light energy is converted into chemical bond energy )photosynthesis
In mitochondria; sugar energy is changed to chemical bond energy of*
.ATP
.The energy of ATP is converted in muscle to mechanical energy*
)5:Enzymes cannot alter reaction equilibria
k1 ]B[
A B )k= velocity constant )mol/sec 100 =
k2 ]A[
:No enzyme k1=10 -4 k2=10 -6 Therefore enzymes accelerate the attainment of equilibria but
:With enzyme k1=10 4 k2=10 2 .do not change it
.B. The active site is a three dimensional entity. It is composed from amino acids that are far apart in the sequence
.D) Binding sites are clefts. Great specificity. Only molecules that contribute to catalysis will fit in the cleft
)Two theories to explain the specificities: 1) the lock and key theory by Emil Fhisher )1894
Energy changes during the reaction: The free energy of the system
.is plotted against the progress of the reaction No Enzyme
Ground states.: The starting point for either forward or reverse reaction
The equilibrium between S and P reflects the difference in the free energies
of their ground states. If P is lower than S, ∆G is negative and the equilibrium
).Favors P )the concentration of P in equilibrium is higher than S
The rate of the reaction is dependent on energy barrier that is required
For the alignment of reacting groups, formation of transient unstable charges
.Bond rearrangements that occurs in the transition state
Transition state: The top of the energy hill at which decay to S or P states is Enzyme
.)Equally probable. The transition state is not a chemical species )like ES or EP
.It is a thermodynamic description of the reaction
The difference between the energy levels of the ground state and the transition states is called.activation energy
The higher the activation energy, the slower the reaction. The rate of a reaction can be increased by raising the
.Temperature, therefore increasing the number of molecules with sufficient energy to overcome the barrier
.Alternatively, the activation energy can be lowered by a catalyst
Therefore, enzymes increase the rate of reactions by lowering the activation states of the reactions
Many reactions have several steps involving the formation and decay of
transient chemical species calledreaction intermediates . The ES and EP
.Complexes are intermediates. They occupy valleys in the reaction diagram
In a multistep reaction the overall rate is determined by the step with the
. highest activation energy-therate limiting step
??How an enzyme lowers the activation energy of a reaction
V=k2 ]]ES
The maximal velocity of a reaction )Vmax) occurs when
:All the enzyme is in a complex with the substrate ES[ is not a measurable concentration. We can[
Measure substrate and the total concentrations
Vmax=k2E t Of the enzyme. Therefore, to find the V of a
Reaction, we have to have a formula that
.use these concentrations
t ]=]E[+]ESE[[
d]ES[ =0
dt
If all the constants are put in
One side of the equation, we can
Combine all of them to one
.Constant, Michaelis constant Km
In such case: ]ES[=]]E[ ]S If we put in the equation ]E[=]Et[ +]ES[ we get:]ES[=]Et[ ]S[ –]]ES[ ]S
Km Km Km
In very low concentrations of substrate ]S[ <<Km then we can neglect S and V= )2]Vmax ]S
Km
.)In small concentrations of S the reaction rate is directly dependent on ]S[ )first order reaction
Kmis often associated with the affinity of enzyme with the substrate. This is true when k cat is a relative small value
Km=k -1+ kcat However, a high value of kcat will affect a high value of Km and in this case Km will
k1 .Not reflect the affinity
kcat .)Gives a direct measure of the catalytic production of product under optimum conditions )saturated enzyme
The unit is sec-1 -known also as theturnover number : the number of substrate molecules turned over per
.Enzyme molecule per second
The ratio k cat/ K Mis convenient measure of enzyme efficiency. We can understand this relation better if we consider
A situation of very low substrate concentration. ]S[<<KM . In such case most of the enzyme is free ]E[t.]~]E
The equation becomes: V=kcat ]]E[]S
KM
kcat / KM . behaves as a second order rate constant for a reaction between substrate and free enzyme
.This ratio provides a direct measure for enzyme efficiency and specificity
The value of kcat/ K Mcan give
A measure for the efficiency
.And specificity of enzymes
Reversible inhibition
:There are different modes of inhibitions that differ in the mechanism by which they decrease enzymes activity
:Competitive inhibition
A molecule resembles the substrate that can compete with the substrate on binding to the active site. The molecule
.Cannot undergo the catalytic step and therefore called an competitive inhibitor
V max
I- I+
KM KM app ]S[
When a molecule can bind to a second site on the enzyme surface in such a way that it modifies kcat. It can for example
distort the enzyme so the catalytic process is not efficient )Allosteric effect). The inhibitor is this case does not
resemble the substrate. The simplest case to consider is one in which the inhibitor does not interfere in any way with
.the substrate binding but completely prevents the catalytic step
KM
The apparent V maxdepends at the concentration
Of I: V max decreases as the concentration of
.I increases
Uncompetitive inhibition
V max
I-
Vmaxapp
I+
V
KM app KM ]S[
Irreversible Inhibition
Many of these inhibitors are similar to the tetrahedral transmission state of enzymes with their substrates and therefore
.Bind with great affinity
Sarin that is known as
Nerve gas binds to serine
In the active site of Acetyl
Cholinesterase and blocks
.Synaptic transmission
TPCK is an inhibitor of
Chemotrypsin. The phenyl
Group fits into the active
Site and position the Cl
To react with imidasole
.Group of His 57
Penicillin inhibits
Glycopeptide transpeptidase
That forms cross links in the
Bacterial cell wall. Covalently
.Binds to Ser in active site
Enzyme activity is affected by pH
Pepsin which
Hydrolyzes peptide
.Enzymes have an optimum pH range for their activity Bonds in the stomach
Has an optimum of
.About 1.6
.Amino acid side chain in the active site may act as weak acids and bases
.It is critical for the catalytic function to maintain state of ionization
Ionic interactions are also critical to maintain the right structure of
.The enzyme
Glucose 6 phosphatase
The optimal pH range of an enzyme helps sometime to know what amino Functions in the liver
Acid is involved. A change in the activity around pH 7 suggest that His is Has an optimum of
.involved responsible ,7.8
To release glucose
.To the blood
Examples for Enzymatic mechanisms of catalysis
The specificity is
Achieved by the
Structure of the
Pocket. In the case
Of trypsin there
Is a negatively
Charged amino acid
.At the pocket
In Chemotrypsin
The pocket contains
Hydrophobic amino
.Acids
.The three dimensional structure of all serine proteases is very similar; evolutionary conserved
.The catalytic domain in this group is composed of three critical amino acids: Ser, His, Asp
The active site can bind also water instead of glucose )OH of water resembles OH of C6), yet the enzyme discriminates
.between glucose and water and prefers glucose by factor of 106
When glucose )and not water) binds it induces a conformational change in the enzyme-induced fit. The binding
energy derived from the interaction induces a conformational change that allows the formation of the active catalytic site
The two step reaction demonstrates a metal ion catalysis and provides an example of general acid-base catalysis
.And transition state stabilization
.Enzymes has to be catalytically active in the right time and in the right place
:Enzymatic activity is regulated in four principle ways
Allosteric interactions. The activity of many multiple subunit )1
,enzymes is regulated by the binding of substrates
.Inhibitors, or activators that induce conformational changes
Later we will discuss the example of aspartate
Transcarbamoylase, an enzyme that is part of the biosynthesis
.of pyrimidines
Reversal covalent modifications: Phosphorylation of certain amino acids in enzymes that is catalyzed by )3
protein kinases. The phosphorylation normally induces conformational changes in the enzymes that will
Either activate or repress their activities. The removal of phosphoryl groups by hydrolysis is catalyzed by
.Protein phosphatases
The concerted model: The binding of substrate to one subunit will switch both from T to R
.State. Symmetry is conserved in this model and not in the sequential model
Aspartate transcarbamoylase
A tetrahedral transition
:State in the catalysis
Histidine 134 stabilize the transition state: When the amino group of aspartate attacks the carbonyl group of carbamoyl
Phosphate, the oxygen becomes negatively charged. The protonated form of His 134 )positively charged) stabilize the
.Negative charge
:The binding of substrates )or PALA) induce large changes in the structure of ATCase
The sequential model predicts that the fraction of catalytic chains in the R state )fR) is equal to the fraction containing
.)Bound substrate )Y
The concerted model predicts that f R .increases more rapidly than Y
.)Binding of nitromethane to tyrosine in the active group forms nitrotyrosine group that is colored )absorbs at 430nm
An essential lysine in the active
Group was modified to block
.Binding of the substrate
.Formed an hybrid enzyme
Catalytic labeled trimers
Were mixed with native trimers
.That can bind the substrate
Adding of succinate changed the
Absorption spectrum of nitrotyrosine
Thus, binding to one trimer changed
.The structure of the other trimer
Allosteric activator shifts the conformational
Equilibrium of all subunits to the R state, whereas
.Inhibitor shifts it towards the T state
Normal regulatory subunits were mixed with
.Nitrotyrosine containing catalytic subunits
Addition of ATP in the absence of substrate increased
The absorbance at 430 nm )like succinate). Thus
.ATP shifted the equilibrium to the R state
CTP decreased the absorbance at 430 nm thus this inhibitor
.Shifted the equilibrium to the T state
Phosphorylation is an effective mean to switch the activity of enzymes
Phosphatases reverse the effect of Kinases are enzymes that transfer the
Kinases by catalyzing the hydrolysis ,gamma phosphoryl group of ATP to serine
Of phosphoryl groups attached to .threonine and tyrosine residues
.The proteins
It adds two negative charges to the protein. Can affect interactions with other protreins and can induce )1
.conformational changes
.The phosphoryl group can form three hydrogen bonds )2
Many hormones trigger the formation of cyclic AMP from ATP. Cyclic AMP
Serves as a messenger in the cell. Its main effect is the activation of protein
Kinase A )PKA). PKA phosphorylates many enzymes on serine and threonine
.Residues
.The enzymes consists of two regulatory )R) subunits and two catalytic )C) subunits
Binding of two molecules of cAMP to each R subunit leads to dissociation of the
.C from the R subunits. And the active site is freed
Proteolytic activation of is another mechanism to activate proteins. Inactive precursors )zymogens) are activated
.By specific cleavage of one or more peptide bonds
.This modification occurs once in the life of an enzyme: it is not reversible
.It can happen also outside cells because it dose not require ATP as phosphorylation of proteins requires
:Biological systems that use proteolytic cleavage are
Digestive enzymes in the stomach and pancreas )1
Some protein hormones like insulin that is synthesized as proinsulin. Insulin is derived by proteolytic removal )3
.Of a peptide
.Fibrous proteins collagen, the major constituent of skin and bones is derived from procollagen, a soluble precursor )4
Developmental processes: The metamorphosis of tadpole into a frog. Large amounts of procollagen are reabsorbed )5
.From the tail and the conversion to the active collagen occurs in a timely and precise mechanism
:Chemotrypsinogen is activated by a specific cleavage of a single peptide bond
.Because the activation step is irreversible, a different mechanism is needed to stop proteolysis
-Specific protease inhibitors, like pancreatic trypsin inhibitor bind very tightly to the active site of trypsin
.)One of the highest affinities in nature )0.1 pM
Antitrypsin is an plasmatic inhibitor of elastase. Elastase is a product of Neutrophils )white blood cells) . Like pancreatic
Trypsin inhibitor,α antitrypson binds almost irreversibly to the active site of elastase. This inhibitor is of extreme
importance. A lys )53) for Glu mutation slows the secretion of this inhibitor from liver cells. People carrying
Homozygous mutation have a disorder calledemphysema . Elestase destroys alveolar walls in the lung by digesting
.Elastic fibers. People with emphysema breath much harder than normal
.Cigarette smoking increase the chances of Heterozygotes to develop emphysema
.The smoke oxidizes Met 358 of the inhibitor
.This methionine is an essential residue for binding elastase
Activation by cleavage- Blood clotting
A cascade of enzymes: The active form of one clotting factor catalyze the activation
Of the next. The many steps yield a large amplification, assuring a rapid response to
.Trauma
Two pathways: the intrinsic pathway is activated from non physiological surface like
.Glass that cause clotting
The Extrinsic pathway is activated by substances that are released from tissues as a
.consequence of trauma
.The final steps of both are common as they cause proper blood clotting
The peptides that are released carry highly negative charges that
Keeps fibrinogen molecules apart. The release of these peptides by
Thrombin change the charges of the central globular region to
Be more positive and the extreme globular regions that carry a
Negative charge can interact with the central region. The newly
Formed clot is stabilized of amid bonds between side chains of
.Glutamine and side chains of lysines in different monomer units
This cross linking binding is catalyzed by transglutaminase
.)Factor XIII)
Thrombin has a mass of 34 Kd and consists of two chains. The B chain is
.)Similar to trypsin, chemotrypsin and elastase )belong to the serine proteases
-It is synthesized as a zimogen called prothrombin. Cleavage between Arg 274
Thr 275 release 32 Kd fragment from the zymogen. Cleavage of Arg 323-Ile 324
Yields the active thrombin. Like in chemotrypsin, an ion pair between the positively
Charged amino group of Ile and a negatively charged group form the active site of
.Thrombin
Therapy: In the past , hemophiliacs were treated by transfusions of concentrated plasma fraction containing
.)VIII. This carried risks of infection )HIV, HBV
The gene was cloned . The human gene of 186 Kb was transfected to the genome of Hamster cells and large
.Amounts of the protein were purified
. The cloning of the gene helps also in prenatal screening for hemophilia mutations
Fibrin Clots are lyzed by plasmin : Plasmin is a serine protease that hydrolizes peptide bonds in the triple stranded
.Connector rode regions of fibrin. Plasmin is formed by proteolytic activation of plasminogen, the inactive precursor
.The conversion is carried by plasminogen activator )TPA). TPA has several domains