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ENZYMES AS CATALYSTS
ROLE OF COENZYMES AND METALS
IN ENZYME CATALYSIS
Associate Professor Ana Savic Radojevic
ENZYMES
There are two fundamental conditions for life:
1.
the living entity must be able to self-replicate
2.
the organism must be able to catalyze chemical reactions efficiently and
selectively
Without catalysis, chemical reactions could not occur on a useful time scale, and thus
could not sustain life.
The conversion of sucrose to CO2 and H2O in the presence of oxygen is a highly
exergonic process, releasing free energy that we can use to think, move, taste, and
see. However, a bag of sugar can remain on the shelf for years without any
obvious conversion to CO2 and H2O. Yet when sucrose is consumed by a human, it
releases its chemical energy in seconds.
The difference is catalysis.
ENZYMES, THE MOST REMARKABLE AND HIGHLY SPECIALIZED PROTEINS
Enzymes have extraordinary catalytic power:
 high degree of specificity for their substrates,
 accelerate chemical reactions tremendously,
 function in aqueous solutions under very mild conditions of temperature and pH.
Enzymes are central to every biochemical process; through the action of regulatory
enzymes, metabolic pathways are highly coordinated.
The study of enzymes has immense practical importance:
 In some genetic disorders, there may be a deficiency or even a total absence of one or
more enzymes.
 Measurements of the activities of enzymes in blood plasma, erythrocytes, or tissue
samples are important in diagnosing certain illnesses.
 Many drugs exert their biological effects through interactions with enzymes.
Much of the history of biochemistry
is the history of enzyme
research
 Biological catalysis was first recognized and described in the late 1700s,
in studies on the digestion of meat by secretions of the stomach.
 In the 1850s, Louis Pasteur concluded that fermentation of sugar into
alcohol by yeast is catalyzed by “ferments” .
 The isolation and crystallization of urease by James Sumner in 1926
provided a breakthrough in early enzyme studies. Sumner found that
urease crystals consisted entirely of protein, and he postulated that all
enzymes are proteins.
 Haldane made the remarkable suggestion that weak bonding
interactions between an enzyme and its substrate might be used to
catalyze a reaction.
ENZYMES ARE CLASSIFIED BY REACTION TYPE:
INTERNATIONAL CLASSIFICATION OF ENZYMES
 this system divides enzymes into six classes, each with subclasses, based on the
type of reaction catalyzed
Enzyme names

o
o
Most enzyme names end in “ase.” Enzymes usually have both:
a common name and
a systematic classification that includes a name and an Enzyme
Commission (EC) number.
Each enzyme is assigned a four-part classification number, Enzyme Commission number
(E.C. number). The formal systematic name of the enzyme catalyzing the reaction:
is ATP:glucose phosphotransferase, which indicates that it catalyzes the transfer of a
phosphoryl group from ATP to glucose. E.C. number is 2.7.1.1.
(2) denotes the class name (transferase);
(7) the subclass (phosphotransferase);
(1) a phosphotransferase with a hydroxyl group as acceptor;
(1) D-glucose as the phosphoryl group acceptor.
For many enzymes, a trivial name is more commonly used—in this case hexokinase.
How enzymes work
 Enzymes are highly effective catalysts, commonly enhancing reaction rates by a factor
of 105 to 1017.
 Enzyme-catalyzed reactions are characterized by the formation of a complex between
substrate and enzyme (an ES complex).
 Substrate binding occurs in a
pocket on the enzyme called
the ACTIVE SITE.
 The function of enzymes is to
lower the activation energy,
G‡ thereby enhance the
reaction rate. The equilibrium
of a reaction is unaffected by
the enzyme.
THE ACTIVE SITE OF THE ENZYME
Enzyme binding sites:
• Active site
• Allosteric site in allosteric enzymes
Specificity of enzyme: active site
The active site is usually a cleft or crevice in the enzyme formed
by one or more regions of the polypeptide chain. Initially, the
substrate molecules bind to their substrate binding sites, also
called the substrate recognition sites. The three-dimensional
arrangement of binding sites in a crevice of the enzyme allows
the reacting portions of the substrates to approach each other
from the appropriate angles.
The active site :
• Substrate binding site
• Active catalytic site
The substrate binding site overlap in the active catalytic site of
the enzyme, the region of the enzyme where the reaction
occurs.

Enzyme specificity (the enzyme’s ability to react with just one substrate) results from the
three-dimensional arrangement of specific amino acid residues in the enzyme that form
binding sites for the substrates and activate the substrates during the course of the reaction.
INDUCED FIT, a mechanism postulated by Daniel Koshland in 1958 :
1.
Induced fit serves to bring specific functional groups on the enzyme
into the proper position to catalyze the reaction.
2.
The conformational change also permits formation of additional weak
bonding interactions in the transition state.
In either case, the new enzyme conformation has enhanced catalytic
properties.
Induced fit is a common feature of the reversible binding of ligands to
proteins. Induced fit is also important in the interaction of almost every
enzyme with its substrate.
” Induced fit” model for substrate binding
As the substrate binds, enzymes undergo a
conformational change (“induced fit”) that repositions
the side chains of the amino acids in the active site and
increases the number of binding interactions.
The substrate binding site is not a rigid “lock” but rather
a dynamic surface created by the flexible overall threedimensional structure of the enzyme.
Conformational change resulting
from the binding of glucose to hexokinase.
MECHANISM OF CATALYSIS

Catalysts increase the rate of chemical reactions by decreasing the
activation energy (Ea)

Catalysts do not change the concentration of substrates and products at
equilibrium, but they do allow equilibrium to be reached more rapidly

No permanent change in catalysts occurs during the reactions they catalyze

Enzymes are biologycal catalysts . A simple enzymatic reaction might be
written
E + S ↔ ES ↔ EP ↔ E + P
Enzymes affect reaction rates, not equilibria


In the coordinate diagram, the free
energy of the system is plotted
against the progress of the reaction
(the reaction coordinate).

THE GROUND STATE (S OR P):The starting point for
either the forward or the reverse reaction.
TRANSITION STATE: the point at the top of the
energy hill at which decay to the S or P state is
equally probable
The transition state is not a chemical species with
any significant stability and should not be confused
with a reaction intermediate (such as ES or EP). It is
a fleeting molecular moment in which events such
as bond breakage/formation, and charge
development have proceeded to the precise point
at which decay to either substrate or product is
equally likely.
The difference between the energy levels of the
ground state and the transition state is the
ACTIVATION ENERGY, ΔG‡.
“STICKASE”




Chemical reactions of many types take place between substrates and
enzymes’ functional groups (specific amino acid side chains, metal ions,
and coenzymes).
The interaction between substrate and enzyme in this complex is mediated
by the same forces that stabilize protein structure, including hydrogen
bonds and hydrophobic and ionic interactions
Much of the energy required to lower activation energies is derived from
weak, noncovalent interactions between substrate and enzyme.
What really sets enzymes apart from most other catalysts is the formation
of a specific ES complex.
Activation energy and the transition state


The functional groups in the catalytic site of the enzyme activate the
substrates and decrease the energy needed to form the high-energy
intermediate stage of the reaction known as the transition state complex.
Some of the catalytic strategies employed by enzymes are general acidbase catalysis, formation of covalent intermediates, and stabilization of
the transition state.
A few principles explain the catalytic power
and specificity of enzymes
A. The rearrangements of covalent bonds during an enzyme-catalyzed reaction
• Catalytic functional groups on an enzyme may form a transient covalent bond with a
substrate.
• These interactions lower the activation energy by providing an alternative, lowerenergy reaction path.
B. The noncovalent interactions between enzyme and substrate
Formation of each weak interaction in the ES complex is accompanied by release of a small
amount of free energy that provides a degree of stability to the interaction. This energy is
called binding energy, GB. Binding energy is a major source of free energy used by enzymes
to lower the activation energies of reactions:
1. This binding energy contributes to specificity as well as to catalysis.
2. Weak interactions are optimized in the reaction transition state; enzyme active sites
are complementary not to the substrates per se but to the transition states through
which substrates pass as they are converted to products during an enzymatic
reaction.
MECHANISM OF CATALYSIS IN ENZYME REACTIONS

General acid-base catalysis- Many reactions involve the formation of unstable
charge intermediates that tend to break down rapidly to their constituent
reactant. They can be often stabilized by the transfer of protons to or from the
substrate or intermediate to form a species that breaks down more rapidly to
products than to reactants

Covalent catalysis- In this type of catalysis, a transient covalent bond is formed
between the enzyme and the substrate
A - B + X: → A - X + B → A + X: + B

Metal ion catalysis-Metals can participate in catalysis in several ways. Ionic
interactions between an enzyme-bound metal and a substrate can help orient
the substrate for reaction or stabilize charged reaction transition states. They
can also mediate oxidation-reduction reactions by reversible change in the
metal,ions oxidation state
Functional groups on amino acid side chains in the active site
MECHANISM OF GENERAL AND SPECIFIC
ACID-BASE CATALYSIS
Reactants
Without catalysis, unstable
(charged) intermediate breaks
down rapidly to form reactants
Proton donor and
acceptor are any acid
(HA) and any base (B:)
Proton donor and
acceptor is
molecule of water
Products
Amino acids in general acid-base catalysis
LYSOZYME: A COMBINATION OF ENZYME-INDUCED SUBSTRATE STRAIN AND
ACID-BASE CATALYSIS
Hexasaccharide
substrate
Heksasaharid-supstrat
Konformacija
Half-chair
polustolice
conformation
Chair
Konformacija
conformation
stolice
Site
of
Mesto
hydrolysis
hidrolize
Conformation of a suggar residue “D” at
which bond breaking occurs is strained from
the stable chair to the unstable half-chair
conformation upon binding
The concept of substrate strain explains the
role of the enzyme in increasing the rate of
reaction
Mechanism for lysozyme action I

Glutamate 35 acts as a proton donor to the glycosidic bond

Glycosidic bond is cleaved and a carbonium ion is formed (positive charge on
carbon 1)

disaccharide NAG leaves enzyme molecule (product 1)
proizvod
Product
P1
P
1
NAG
NAG
O
E
O
E
H
C4
O
Glu
35
Glu
35
C1
C
O-
O
O
O
O
H
C4
O-
O
C
D
-O
NAG 3
H
C
Asp
52
C1
O
+
O
C
D
-O
NAG 3
Asp
52
Mechanism for lysozyme action II

carbonium ion intermediate reacts with OH-group from water

tetra-NAG (product 2) leaves enzyme molecule

glutamate 35 is reprotonated with H+ from water and enzyme is ready for new
catalysis
O
H
proizvod
Product
P2
P2
H
OGlu
35
O
C
Glu
35
H
O
C1
O
+
C
H
O
O
C
D
-O
NAG 3
Asp
52
H
HO
C1
O
O
C
D
-O
NAG 3
Asp
52
CATALYTIC MECHANISM OF CHYMOTRYPSIN
• Chymotrypsin is a digestive enzyme that catalyzes
the hydrolysis of specific peptide bonds in
denatured proteins. It is a member of the serine
protease superfamily, enzymes that use a serine in
the active site to form a covalent intermediate
during proteolysis.
•In the overall hydrolysis reaction the carbonyl
carbon, which carries a partial positive charge, is
attacked by a hydroxyl group from water. An
unstable tetrahedral oxyanion intermediate is
formed, which is the transition state complex. As
the electrons return to the carbonyl carbon, it
becomes a carboxylic acid, and the remaining
proton from water adds to the leaving group to
form an amine
I stage:cleavage of the peptide bond in the denatured substrate and formation of a
covalent acyl-enzyme intermediate
1. Substrate binding
As the substrate protein binds to
the active site, Ser195 and His57 are
moved closer together for the nitrogen
electrons on His to attract the hydrogen
of Ser. Without this change of
conformation on substrate binding, the
catalytic triad cannot form.
2. Histidine activates serine for nucleophilic attack
His serves as a general base
catalyst as it abstracts a proton
from the Ser, increasing the
nucleophilicity of the serineoxygen, which attacks the carbonyl
carbon.
I stage:cleavage of the peptide bond in the denatured substrate and formation of a
covalent acyl-enzyme intermediate
3. The oxyanion tetrahedral intermediate is
stabilized by hydrogen bonds
The electrons of the carbonyl group
form the oxyanion tetrahedral
intermediate. The oxyanion is stabilized
by the NH groups of Ser195 and glycine
4. cleavage of the peptide bond
The amide nitrogen in the peptide bond
is stabilized by interaction with the His
proton (general acid catalysis). As the
electrons of the peptide bond withdraw
into the nitrogen, the electrons of the
carboxyanion return to the substrate
carbonyl carbon, resulting in cleavage of
the peptide bond.
I stage: cleavage of the peptide bond in the denatured substrate and formation of a
covalent acyl-enzyme intermediate
5. The covalent acyl–enzyme intermediate
The cleavage of the peptide bond
results in formation of the covalent
acyl-enzyme intermediate, and the
amide half of the cleaved protein
dissociates
STAGE II - hydrolysis of the acyl-enzyme intermediate
6. Water attacks the carbonyl carbon
The active site His activates water to form
an OH- for a nucleophilic attack.
7. Second oxyanion tetrahedral intermediate
Second oxyanion transition state is formed.
The oxyanion is again stabilized
by the NH groups of Ser195 and glycine in
the chymotrypsin peptide backbone
STAGE II - hydrolysis of the acyl-enzyme intermediate
8. Acid catalysis breaks the acyl–enzyme
covalent bond
The active site histidine adds the proton
back to serine .
MMMMMMMM
9. The product is free to dissociate
The reaction is complete and the product
dissociates .
Energy diagram in the presence of chymotrypsin
Mechanism-based inhibitors.


The effectiveness of many drugs
and toxins depends on their ability
to inhibit an enzyme.
The strongest inhibitors are
covalent inhibitors, compounds that
form covalent bonds with a reactive
group in the enzyme active site, or
transition state analogues that
mimic the transition state complex.
CATALYTIC RESIDUES ARE
HIGHLY CONSERVED

Members of an enzyme family such as the aspartic or serine proteases employ a similar
mechanism to catalyze a common reaction type but act on different substrates. Most enzyme
families arose through gene duplication events that create a second copy of the gene that
encodes a particular enzyme.

The proteins encoded by the two genes can then evolve independently to recognize different
substrates—resulting, for example, in chymotrypsin, which cleaves peptide bonds on the
carboxyl terminal side of large hydrophobic amino acids, and trypsin, which cleaves peptide
bonds on the carboxyl terminal side of basic amino acids.

Proteins that diverged from a common ancestor are said to be homologous to one another.

The common ancestry of enzymes can be inferred from the presence of specific amino acids in
the same position in each family member. These residues are said to be conserved residues.

Among the most highly conserved residues are those that participate directly in catalysis.
ISOZYMES ARE DISTINCT ENZYME
FORMS THAT CATALYZE THE SAME
REACTION

Higher organisms often elaborate several physically distinct versions of a given enzyme, each
of which catalyzes the same reaction. Like the members of other protein families, these
protein catalysts or isozymes arise through gene duplication. Isozymes may exhibit subtle
differences in properties such as sensitivity to particular regulatory factors or substrate affinity
(eg, hexokinase and glucokinase) that adapt them to specific tissues or circumstances. Some
isozymes may also enhance survival by providing a “backup” copy of an essential enzyme.
Prosthetic groups, cofactors and coenzymes
•
•
Prosthetic groups, cofactors and coenzymes – are small nonprotein
molecules and metal ions that participate directly in substrate binding or
catalysis
Holoenzyme is catalyticly active enzyme with it’s coenzyme or metal ion,
protein part of holoenzyme is apoenzyme
Prosthetic Groups Are Tightly Integrated Into an Enzyme’s Structure

Prosthetic groups are distinguished by their tight, stable incorporation into a
protein’s structure by covalent or noncovalent forces. Examples include pyridoxal
phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD),
thiamine pyrophosphate, biotin, and the metal ions of Co, Cu, Mg, Mn, and Zn.

Metals are the most common prosthetic groups. The roughly one-third of all
enzymes that contain tightly bound metal ions are termed metalloenzymes. Metal
ions that participate in redox reactions generally are complexed to prosthetic
groups such as heme or iron-sulfur clusters.

Metals also may facilitate the binding and orientation of substrates, the formation
of covalent bonds with reaction intermediates (Co2+ in coenzyme B12), or interact
with substrates to render them more electrophilic (electron-poor) or nucleophilic
(electron-rich).
Cofactors Associate Reversibly With Enzymes or Substrates

Cofactors serve functions similar to those of prosthetic groups but bind in a transient,
dissociable manner either to the enzyme or to a substrate such as ATP. Unlike the stably
associated prosthetic groups, cofactors therefore must be present in the medium surrounding
the enzyme for catalysis to occur.

The most common cofactors also are metal ions. Enzymes that require a metal ion cofactor
are termed metal-activated enzymes to distinguish them from the metalloenzymes for
which metal ions serve as prosthetic groups.
Coenzymes Serve as Substrate Shuttles

Coenzymes serve as recyclable shuttles—or group transfer agents—that transport many
substrates from their point of generation to their point of utilization. Association with the

coenzyme also stabilizes substrates such as hydrogen atoms or hydride ions that are unstable
in the aqueous environment of the cell. Other chemical moieties transported by coenzymes

include methyl groups (folates), acyl groups (coenzyme A), and oligosaccharides (dolichol).
Many Coenzymes, Cofactors & Prosthetic
Groups Are Derivatives of B Vitamins

The water-soluble B vitamins supply important components of numerous coenzymes. Several
coenzymes contain, in addition, the adenine, ribose, and phosphoryl moieties of AMP or ADP.
Nicotinamide is a component of the redox coenzymes NAD and NADP, whereas riboflavin is a
component of the redox coenzymes FMN and FAD.

Pantothenic acid is a component of the acyl group carrier coenzyme A.

As its pyrophosphate, thiamin participates in decarboxylation of α-keto acids, and folic acid
and cobamide coenzymes function in one-carbon metabolism.
Some inorganic elements that serve as cofactors for enzymes
Cu2+
cytohrome oxidase
Fe2+ ili Fe3+
cytohrome oxidase, catalase, peroxydase
K+
pyruvate kinase
Mg2+
hexokinase, glucoso 6-phosphatase, pyruvate
kinase
Mn2+
ribonucleotide reductase
Se
glutathione peroxydase
Zn2+
carbonic anhydrase, carboxypeptidase A & B
Some coenzymes that serve as transient carries of specific atoms or
functional groups
Coenzyme
Chemical group for transfer
Vitamin
Biotin
CO2
biotin
Coenzyme A (CoA)
acyl group
pantothenic acid
Coenzyme B12 (CoB12)
H atom / alkyl group
cobalamine(B12)
Flavin adenine
dinucleotide (FAD)
electrons
riboflavin (B2)
Nicotinamide adenine
dinucleotide (NAD)
hydride ion (H-)
niacine
Pyridoxal phosphate
amino group
pyridoxine (B6)
Tetrahydrofolate
one-carbon fragments
folic acid
Thiamine diphosphate
aldehyde
thiamine (B1)
Role of metals in catalysis




Metalloenzymes are enzymes that contain tightly bound metal ions
Sometimes metal ions are part of prosthetic nonprotein group such as heme
Enzymes that require a metal ion cofactor are termed metal-activated
enzymes. In that case metal isn’t integrated into an enzyme’s structure
Role of metals:
A. stabilize active conformation of enzyme;
B. also may facilitate the binding and orientation of substrate;
C. can act as direct catalysts;
D. have an important role in oxido-reduction reactions
Metals cofactors have various functions
• Some transition metals like Zn, Fe, Mn
i Cu can act as Lewis acid, because they
have empty d electron orbitals
• A good example of a metal functioning
as a Lewis acid is found in carbonic
anhydrase (zinc enzyme). The first step
is generation of a proton and a hydroxyl
group which binds to the zinc. The
proton and hydroxyl group are then
added to the carbon dioxide and carbon
acid is relaesed
Role of the metals
Enz-S-M: “Substrate-bridged” complex
1. The true substrate for creatine kinase is
not ATP, but Mg2+-ATP. In this case Mg2+
does not interact directly with the
enzyme, but may serve to neutralize the
negative charge density on ATP and
faciliate binding to the enzyme
2. Scheme for the binding of Mg2+-ATP and
glucose in the active site of hexokinase
3. All kinases exept muscule pyruvate
kinase and phosphoenolpyruvate kinase
are“Substrate-bridged” complexes
Enz-M-S: “Metal-bridged” complex
•
This enzymes contain a tightly bound
transitional metals (Zn ili Fe)
Model of the role of K+ in the active site of pyruvate kinase
phosphoenol-pyruvate +ADP pyruvate kinase ATP + pyruvate
A. It belongs to Enz-M-S bridged
complexes. Mg coordinates the
substrate to the enzyme active site
B. Initial binding
of K+ induces
conformational changes in the kinase,
which results in increased affinity for
phosphoenol-pyruvate. In addition K+
orients the phosphoenol-pyruvate in
the correct position for transfer of it’s
phosphate to ADP, the second substrate
Thus, K+ and Na+, stabilize active
conformation of the enzyme but are
passive in cataysis.
pH and temperature profiles.


Enzymes have a functional pH range determined by the pKa of functional
groups in the active site and the interactions required for threedimensional structure.
Non-denaturing increases of temperature increase the reaction rate.
Catalytic RNAs: RIBOZYMES


Some of the most interesting molecular events in RNA metabolism occur
during this postsynthetic processing. Intriguingly, several of the enzymes
that catalyze these reactions consist of RNA rather than protein.
The discovery of these catalytic RNAs, or ribozymes, has brought a
revolution in thinking about RNA function and about the origin of life.
RNA Catalyzes the Splicing of Introns
Self-splicing group I introns share several properties with enzymes besides accelerating
the reaction rate, including their kinetic behaviors and their specificity. Because the
intron itself is chemically altered during the splicing reaction—its ends are cleaved—it
may appear to lack one key enzymatic property: the ability to catalyze multiple reactions.
Most of the activities of these ribozymes are based on two fundamental reactions:
transesterification and phosphodiester bond hydrolysis (cleavage). The substrate for
ribozymes is often an RNA molecule, and it may even be part of the ribozyme itself.


The enzymatic activity that catalyzes peptide bond formation has historically
been referred to as peptidyl transferase and was widely assumed to be
intrinsic to one or more of the proteins in the large ribosomal subunit.
We now know that this reaction is catalyzed by the 23S rRNA, adding to the
known catalytic repertoire of ribozymes. This discovery has interesting
implications for the evolution of life

Enzyme-Linked Immunoassays

The sensitivity of enzyme assays can be exploited to detect proteins that lack catalytic activity.
Enzyme-linked immunosorbent assays (ELISAs) use antibodies covalently linked to a
“reporter enzyme” such as alkaline phosphatase or horseradish peroxidase whose products
are readily detected, generally by the absorbance of light or by fluorescence.

Serum or other biologic samples to be tested are placed in a plastic microtiter plate, where
the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas
of the well are then “blocked” by adding a nonantigenic protein such as bovine serum
albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The
antibodies adhere to the immobilized antigen and are themselves immobilized.

Excess free antibody molecules are then removed by washing.

The presence and quantity of bound antibody is then determined by adding the substrate for
the reporter enzyme.
A significant part of the energy used for enzymatic rate enhancements is
derived from WEAK INTERACTIONS (hydrogen bonds and hydrophobic and ionic
interactions) between substrate and enzyme.
Enzymes are highly effective catalysts, commonly enhancing reaction
rates by a factor of 105 to 1017.
Some of these weak interactions occur preferentially in the reaction
transition state, thus stabilizing the transition state.
Enzyme-catalyzed reactions are characterized by the formation of an
ES complex. Substrate binding occurs in a pocket on the enzyme called
the active site
The binding energy, GB, can be used to lower substrate entropy or to
cause a conformational change in the enzyme (INDUCED FIT). Binding
energy also accounts for the exquisite specificity of enzymes for their
substrates.
Additional catalytic mechanisms include GENERAL ACID-BASE CATALYSIS,
COVALENT CATALYSIS, AND METAL ION CATALYSIS. Catalysis often involves
transient covalent interactions between the substrate and the enzyme, or
group transfers to and from the enzyme to provide a new, lower-energy
reaction path