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Structural Biochemistry/Enzyme
Structural Biochemistry/Enzyme
Overview
Enzymes are macromolecules that help accelerate (catalyze) chemical reactions in biological systems. Some
biological reactions in the absence of enzymes may be as much as a million times slower. Virtually all enzymes are
proteins, though the converse is not true and other molecules such as RNA can also catalyze reactions. The most
remarkable characteristics of enzymes are their ability to accelerate chemical reactions and their specificity for a
particular substrate. Enzymes take advantage of the full range of intermolecular forces (van der waals interactions,
polar interactions, hydrophobic interactions and hydrogen bonding) to bring substrates together in most optimal
orientation so that reaction will occur. Also, enzymes can be inhibited by specific molecules by called competitive,
uncompetitive, and noncompetitive inhibitors.
Catalysis happens at the active site of the enzyme. It contains the residues that directly participate in the making and
breaking of bonds. These residues are called the catalytic groups. Although enzymes differ widely in structure,
specificity, and mode of catalysis a number of generalizations concerning their active sites can be made:
1. The active site is a three dimensional cleft or crevice formed by groups that come from different parts of the
amino acid sequence - residues far apart in the amino acid dequence may interact more strongly than adjacent
residues in the sequence.
2. The active site takes up a relatively small part of the total volume of an enzyme. Most of the amino acid residues
in an enzyme are not in contact with the substrate, which raises the question of why enzymes are so big. Nearly all
enzymes are made up of more than 100 amino acid residues. The "extra" amino acids serve as a scaffold to creat the
three dimensional active site from the amino acids that are far apart in the primary structure. In many proteins the
remaining amino acids also constitute regulatory sites, sites of interaction with other proteins, or channels to bring
the substrate to the active sites.
3. Active sites are unique microenvironments. In all enzymes of known structure, substrate molecules are bound to a
cleft or crevice. Water is usually excluded unless it is a reactant. The nonpolar microenvironment of the cleft
enhances the binding of substrates as well as catalysis. Nevertheless, the cleft may also contain polar residues.
Certain of these polar residues acquire special properties essential for substrate bidning or catalyis.
4. Substrates are bound to enzymes by multiple weak interations. Stated above
5. The specificity of binding depends on the precise defined arrangement of atoms in the active site. Because the
enzyme and the substrate interact by means of short-range forces that require close contact, a substrate must have a
matching shape to fit into the site. However, the active site of some enzymes assume a shape that is complementary
to that of the substrate only after the substrate is bound. This process of dynamic recognition is called induced fit.
Enzymes are highly specific and may require cofactors for catalysis. A cofactor is a non-protein chemical compound
bound to a protein; there are 2 types of cofactors: Metals and organic/metalloorganic (which are derived from
vitamins). An example of a metal cofactor is zinc and the enzyme, carbonic anhydrase, tightly binds the zinc at the
active site. The process involves binding water to carbon dioxide and deprotonating it into carbonic acid. Then the
carbonic acid becomes a bicarbonate ion due to the displacement of water.
Catalysts can fasten the reaction speed by lowering the activation energy (not the transition state) of the process. The
active site is a location on the enzyme which has complementary shape to the substrate. It is also where the amino
acids with a complementary charge, polarity and shape to the ligand are.
The enzyme function and catalysis result from the ability to stabilize the transition state in a chemical reaction. The
transition state is the highest energy species in a reaction. It is a transitory molecular structure that is no longer the
substrate but is not yet the product. It is the most seldom occupied species along the reaction pathway. The
difference in free energy between the transition state and the substrate is called the Gibbs free energy of activation or
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simply the activation energy.
Thus we can see the key to how enzymes operate: Enzymes accelerate reactions by decreasing the activation energy.
The combination of substrate and enzyme creates a reaction pathway whose transition state is lower than that of the
reaction in the absence of the enzyme. Because the activation energy is lower more substrate molecules have the
energy required to reach the transition state.
It is important to note that enzymes have evolved specifically to recognize the transition states of chemical reactions.
Therefore, enzymes do not bind to any reactive species before the species have actually begun to react; enzymes only
recognize and bind the transition states of such species. In fact, if enzymes were to bind to the reactants of a reaction
"on sight", or immediately, this would result in an even higher activation energy than before! For this reason,
enzymes recognize only the transition state and bind to reactive species only when this high-energy state has been
achieved. The fact that enzymes can recognize structures as specific and short-lived as transition states is a testament
to their incredible specificity and efficiency.
Each enzyme is optimized for a particular reaction transition state. This ensures that enzymes will not compete with
each other and hinder cellular reactions instead of help them. Enzyme inhibition occurs when the activity of a given
enzyme is disrupted or interrupted in some fashion. Inhibitors can be molecules that have a similar shape, structure,
or charge to the substrate in question so that the active site of an enzyme will "mistake" the inhibitor for the
substrate. This affects the affinity of the enzyme for the substrate, as well as the rate of the overall reaction. Several
types of inhibition can occur in the cell; more detailed explanations on these can be found in the corresponding
sections.
Because of the active sites, enzymes are highly specific catalysts. These catalysts are governed by the ability to
lower the free energy of thermodynamics to overcome transition states. The Michaelis-Menten Model describes the
kinetic properties of many enzymes.
The interaction between the substrate and the enzyme helps accelerate the reaction, and the specificity of enzymes
result in minimal side reactions.
It is of great importance to note that an enzyme cannot alter the laws of thermodynamics and consequently cannot
alter the equilibrium of the reaction. The amount of product formed for a reaction utilizing an enzyme is always
equal to the amount of product form of the same reaction occuring in the same reaction mixture without the enzyme.
The enzyme just allows the reaction to reach its equilibrium faster. The equilibrium position is a function only of the
free-energy difference between reactants and products.
Lock and Key Model
The "lock and key" model was first proposed by an organic chemist named Emil Fischer in 1894. In this model, the
"lock" refers to an enzyme and the "key" refers to its complementary substrate. Each enzyme has a highly specific
geometric shape that is complementary to its substrate. In order to activate an enzyme, its substrate must first bind to
the active site on the enzyme. Only then will a catalytic reaction take place. However, like a lock and a key, the
enzyme and substrate shape must be complementary and fit perfectly. Designed by evolution the active site for
enzymes is generally highly specific in its substrate recognition and has the ability to distinguish between
sterioisomers.
Induced Fit
According to the Lock and Key Model, the geometric shape of both enzymes and substrates can not be changed as
they are both predetermined. Thus, the binding of the substrate to the enzymes active site does not alter the shape of
the enzyme. While this theory helped explain the specificity of the enzyme, it does not explain the stability of the
transition state for it would require more energy to reach the transition state complex. Thus the induced fit model
was proposed in which enzymes like proteins are flexible. The concept of induced fit is that when a substrate binds
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to the active site of an enzyme, there is a conformational change and structural adaptation that makes this binding
site more complementary and tighter. In essence the substrate does not simply bind to a rigid active site but instead
the macromolecules, weak interaction forces, and hydrophobic characteristics on the enzyme surface mold into a
precise formation so that there is an induced fit where the enzyme can perform maximum catalytic function.
Transition State Theory
Transition state theory states that in an enzyme
catalysis, the enzyme binds more strongly to its
"transition state complex rather than its ground state
reactants." In essence, the transition state is more
stable. The stabilization of the transition state lowers
the activation barrier between reactants and products
thus increasing the rate of reaction or enzymatic
activity as this will favor the increase of formation of
the transition state complex.
In the transition state theory, the mechanism of
interaction of reactants is irrelevant. However, the
Stabilization of the transition state by an enzyme.
colliding molecules that take place in the reaction must
have sufficient amount of kinetic energy to overcome the activation energy barrier in order to react. In many cases,
temperature, pH, or enzymes can be changed to facilitate the stabilization of the transition state as well as
statistically increasing the probability for molecules colliding and forming the transition state complex. For a
bimolecular reaction such as Sn2, a transition state is formed when the two molecules’ old bonds are weakened and
new bonds begin to form or the old bonds break first to form the transition state and then the new bonds form after.
The theory suggests that as reactant molecules approach each other closely they are momentarily in a less stable state
than either the reactants or the products.
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Methods
1. Some catalysts provide a charge to a molecule to make it more attractive to other reactants. Acids are an example
for this kind of catalyst. They give the reacting species a positive charge to attract the negative or partially
negative reactant, increasing the chance for the two species to collide and react.
2. Some catalysts increase the local concentration of reactants so that they are more likely to collide.
3. Some catalysts may modify the shape of one reactant to be more susceptible to other molecule.
Enzymatic Strategies and Examples
1. Covalent Catalysis - Through the course of catalysis, a powerful nucleophile is temporarily attached to a part of
the substrate. The nucleophile is contained in the active site. A proteolytic enzyme chymotrypsin is an excellent
example of this strategy.
2. General Acid/Base Catalysis - Water often acts as a donor or acceptor, but in Acid/base catalysis, the molecule
which donates or accepts a proton is NOT water. This strategy incorporates base and acid catalysis to shorten
reaction times. In the case of Chymotrypsin, the enzyme uses a histidine residue as a base catalyst to enhance the
nucloephilicity of serine analogous to how hisitidne residue in carbonic anhydrase facilitates the removal of a proton
from a zinc bound water molecule to yield hydroxide.
3. Catalysis by approximation - This method involves reactions where the molecule react with two substrates. The
two substrates are brought together to one area and this increases the rate of the reaction. NMP kinase for example,
brings tow nucleotides together to improve the transferring of phosphoryl groups.
4. Metal Ion Catalysis - Metal ions can be involved as a catalyst in many different ways. Zinc can help the formation
of a nucleophile. It makes the pka of water change from approximately 14 to 7, which allows it to be protonated at
neutral pH. It can also stabilize negative charges by acting as an electrophile in a complex. Metal ions are also used
to increase the binding energy of substrates, holding them together. A metal ion may also serve as a bridge between
the enzyme and substrate acting as a cofactor in cases of NMP kinases.
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Enzyme's Cofactors for Activity
The catalytic activity of enzymes depends
on the presence of small molecules called
cofactors. The role of the catalytic activity
varies with the enzyme and its cofactors. In
general, those cofactors can execute
chemical reactions which cannot be
performed by the standard 20 amino acids.
An enzyme without cofactor is called
apoenzyme, however the one with
completely catalytically active is called
holoenzyme.
Cofactors can be divided into two individual
groups: Metal and Coenzymes. Metals are
important for enzymes because they are
molecular assistants that play a vital role in
some of the enzymatic reactions that fuel the
body metabolism. They also act to stabilize
the shapes of enzymes. For example, iron
helps the protein hemoglobin transport
The succinate dehydrogenase complex showing several cofactors, including flavin,
oxygen to organs in the body and copper
iron-sulfur centers and heme.
helps superoxide dismutase in sopping up
dangerous free radicals that accumulate inside the cells. Coenzymes are small organic molecules that often derived
from vitamins. Coenzymes can be either tightly or loosely bound to the enzyme. Tightly bound ones are called
prosthetic groups, while loosely bound coenzymes are like substrates and products, bind to the enzyme and get
released from it. Enzymes that use the same coenzymes often perform catalysis by the similar mechanisms.
Enzyme Classification
Class
Type of Reduction
Examples
Hydrolases
Catalyze hydrolysis reactions
Estrases Digestive enzymes
Isomerases
Catalyze isomerization (changing of a molecule into its isomer)
Phospho hexo isomerase, Fumarase
Ligases
Catalyze bond formation coupled with ATP hydrolysis.
Citric acid synthetase
Lyases
Catalyze a group elimination in order to form double bonds (or a ring structure). Decarboxylases Aldolases
Oxidoreductases Catalyze oxidation-reduction reactions
Dehydrogenases Oxidases
Transferases
Transaminase Kinases
Catalyze the transfer of functional groups among molecules.
The classification of an enzyme is shown within the table as it's class and the type of reduction the enzyme goes
through. An example of a name is glucose phosphotransferase. In this reaction ATP transfers one of it's phosphates
to glucose: ATP + D-glucose -> ADP + D-glucose 6-phosphate. Since this process "transfers" a phosphate group to
glucose, it is within the classification of transferases, hence the name "glucose phosphotransferase." Since many
enzymes have common names that do not refer to their function or what kind of reaction they catalyze, a enzyme
classification system was established. There are six classes of enzymes that were created with subclasses based on
what they catalyze so that enzymes could easily be named. Depending on the type of reaction catalyzed, an enzyme
can have various names. These classes are Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, and
Structural Biochemistry/Enzyme
Ligases. This is the internation classification used for enzymes. For example, a common oxidoreductase is
dehydrogenase. Dehydrogenase is known as an enzyme that oxidizes a substrate and transferring protons. Enzymes
are normally used for catalyzing the transfer of functional groups, electrons, or atoms. Since this is the case, they are
assigned names by the type of reaction they catalyze. This allowed for the addition of a four-digit number that would
precede EC(Enzyme Commission) and each enzyme could be identified. The reaction that an enzyme catalyzes must
be know before it can be classified.
Oxidoreductases catalyze oxidation-reduction reactions where electrons are transferred. These electrons are usually
in the form of hydride ions or hydrogen atoms. When a substrate is being oxidized it is the hydrogen donor. The
most common name used is a dehydrogenase and sometimes reductase will be used. An oxidase is referred to when
the oxygen atom is the acceptor.
Transferases catalyze group transfer reactions. The transfer occurs from one molecule that will be the donor to
another molecule that will be the acceptor. Most of the time, the donor is a cofactor that is charged with the group
about to be transferred.
Hydrolases catalyze reactions that involve hydrolysis. This cases usually involves the transfer of functional groups to
water. When the hydrolase acts on amide, glycosyl, peptide, ester, or other bonds, they not only catalyze the
hydrolytic removal of a group from the substrate but also a transfer of the group to an acceptor compound. These
enzymes could also be classified under transferaes since hydrolysis can be viewed as a transfer of a functional group
to water as an acceptor. However, as the acceptor's reaction with water was discovered very early, it's considered the
main function of the enzyme which allows it to fall under this classification.
Lyases catalyze reactions where functional groups are added to break double bonds in molecules or the reverse
where double bonds are formed by the removal of functional groups.
Isomerases catalyze reactions that transfer functional groups within a molecule so that isomeric forms are produced.
These enzymes allow for structural or geometric changes within a compound. Sometime the interconverstion is
carried out by an intramolecular oxidoreduction. In this case, one molecule is both the hydrogen acceptor and donor,
so there's no oxidized product. The lack of a oxidized product is the reason this enzyme falls under this classification.
The subclasses are created under this category by the type of isomerism.
Ligases are used in catalysis where two substrates are litigated and the formation of carbon-carbon, carbon-sulfide,
carbon-nitrogen, and carbon-oxygen bonds due to condensation reactions. These reactions are couple to the cleavage
of ATP.
The Michaelis-Menten Model
The Michaelis-Menten model is used to describe the kinetic properties of many enzymes. In this model, an
enzyme(E)combines with a substrate(S)to form an enzyme-substrate(ES)complex, and proceed to form a
product(P)or to dissociate into E and S.
The rate of formation of product,V0, can be calculated by the Michaelis-Menten equation:
Vmax is the reaction rate when the enzyme is completely saturated with substrate. KM is the Michaelis constant,
which is the substrate concentration at the half of the maximum reaction rate. The kinetic constant kcat isa called the
turnover number, which is the number of substrate molecules converted into produce per unit time at a single
catalytic site when the enzyme is saturated with substrate. It often count for most enzyme between 1 and 104per
second.
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Structural Biochemistry/Enzyme
Allosteric enzymes is an important class of enzymes. Its catalytic activity can be regulated. It has multiple active
sites which display cooperativity, as evidenced by a sigmoidal dependence of reaction velocity on substrate
concentration. We also find that K max is the substrate concentration in which the overall reaction rate at that
particular time is half of V max. V max on the other hand, is the maximum reaction rate in which the active site is
completely saturated with substrate. As a result of this physical characteristic, we see that no matter how much
substrate is consequently added, the relative rate of the reaction remains unchanged as additional substrate do not
contribute to any kinetic interaction with binding the active site. The affinity also eventually does not change as more
substrate is increased and the reaction goes towards equilibrium.
Replicative DNA polymerase
There have been studies of the three multi-subunit DNA polymerase enzymes in the nucleus. This provides insights
into the makeup of the replication machinery in eukaryotic cells. The first DNA polymerase structure to by solved
crystallographically was the Klenow fragment of E. coli DNA polymerase I. This crystallization revealed a structure
that was likened to the palm, fingers, and thumb of a right hand. Studies of the Klenow fragment showed that DNA
was bound within the cleft and that the fingers and thumb architecture is conserved in many of the polymerase
families. The polymerase active site residues are located in the palm domain. The fingers are important for
nucleotide binding, and the thumb domain binds the DNA.
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References
http:/ / www. tutorvista. com/ content/ biology/ biology-iii/
cellular-macromolecules/enzymes-classification.php
DNA polymerases adds nucleotides to the 5' end
of a strand of DNA <Allison, Lizabeth A.>
<Allison, Lizabeth A. Fundamental Molecular
Biology. Blackwell Publishing. 2007. p.112>. If a
mismatch is accidentally incorporated, the
polymerase is inhibited from further extension.
Proofreading removes the mismatched nucleotide
and extension continues.
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