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Enzymes
2016
Enzymes are biologic polymers that catalyze the chemical reactions by increasing
the rate of reactions without being changed in the overall process.
• substance that increase rates of a chemical reaction
• does not effect equilibrium
• remain unchanged in overall process
• reactants bind to catalyst, products are released
Biomedical importance:
• Enzymes play an important role in metabolism, digestion and therapeutics.
• The presence and maintenance of enzymes is essential for the breakdown of
nutrients to supply energy and chemical building blocks
• With the exception of catalytic RNA molecules, or ribozymes, enzymes are
proteins.
• The ability to assay the activity of specific enzymes in blood, other tissue
fluids, or cell extracts aids in the diagnosis and prognosis of disease.
• Deficiencies in the quantity or catalytic activity of key enzymes can result from
genetic defects, nutritional deficits, or toxins.
Medical scientists address imbalances in enzyme activity by using pharmacologic
agents to inhibit specific enzymes and are investigating gene therapy as a means
to remedy deficits in enzyme level or function.
• Proteolytic enzymes augment the capacity of detergents to remove dirt and
stains.
Enzymes play an important role in producing or enhancing the nutrient value of
food products for both humans and animals. The protease rennin, for example, is
utilized in the production of cheeses while lactase is employed to remove lactose
from milk for the benefit of persons who suffer from lactose intolerance as a
consequence of a deficiency in this hydrolytic enzyme
Nomenclature of enzymes :
• A. Recommended name
• Most commonly used enzyme names have the suffix "-ase" attached to the
substrate of the reaction (for example, glucosidase urease, sucrase), or to a
description of the action performed (for example, lactate dehydrogenase and
adenylyl cyclase). [Note: Some enzymes retain their original trivial names,
which give no hint of the associated enzymic reaction, for example, trypsin and
pepsin.]
B. Systematic name (Classification of enzymes)
• The International Union of Biochemists (IUB) developed a system of enzyme
nomenclature in which each enzyme has a unique name and code number
that identify the type of reaction catalyzed and the substrates involved.
• Enzymes are divided into six major classes , each with numerous subgroups.
Classification of enzymes
• 1. Oxidoreductases (catalyze oxidations and reductions)
• 2. Transferases (catalyze transfer of moieties such as glycosyl, methyl, or
phosphoryl groups)
• 3. Hydrolases (catalyze hydrolytic cleavage of C—C, C—O, C—N, and other
bonds)
• 4. Lyases (catalyze cleavage of C—C, C—O, C—N, and other bonds by atom
elimination, leaving double bonds)
• 5. Isomerases (catalyze geometric or structural changes within a molecule)
• 6. Ligases (catalyze the joining together of two molecules coupled to the
hydrolysis of ATP)
•
Properties of enzymes :
• Enzymes are protein catalysts that increase the velocity of a chemical reaction,
and are not consumed during the reaction they catalyze. [Note: Some types of
RNA can act like enzymes, usually catalyzing the cleavage and synthesis of
phosphodiester bonds. RNAs with catalytic activity are called ribozymes , and
are much less commonly encountered than protein catalysts.]
• A. Active sites
• Enzyme molecules contain a special pocket or cleft called
the active site. The active site contains amino acid side chains
that create a three-dimensional surface complementary to
the substrate (Figure 5.2). The active site binds the substrate,
forming an enzyme-substrate (ES) complex. ES is converted
to enzyme-product (EP), which subsequently dissociates to
enzyme and product.
Properties of enzymes :
B. Catalytic efficiency
Most enzyme-catalyzed reactions are highly efficient, proceeding from 103 to 1016 times
faster than un catalyzed reactions. Typically, each enzyme molecule is capable of
transforming 100 to 1000 substrate molecules into product each second.
Turnover number :The number of molecules of substrate converted to product per
enzyme molecule per second.
• Urease is a good example:
– Catalyzed rate: 3x104/sec
– Uncatalyzed rate: 3x10 -10/sec
– Ratio is 1x1014 !
C. Specificity
Enzymes are highly specific, interacting with one or a few substrates and catalyzing only
one type of chemical reaction.
Properties of enzymes :
Holoenzyme refers to the enzyme with its cofactor (active enzyme).
Apoenzyme refers to the enzyme without its non protein portion of the
holoenzyme and its inactive.
Holoenzyme = Apoenzyme + non protein portion (Prosthetic groups , Co enzyme)
D. Prosthetic groups
Prosthetic groups are distinguished by their tight, stable incorporation into a
protein's structure by covalent or non covalent forces for ( example, the biotin
bound to carboxylases and the metal ions of Co, Cu, Mg, Mn, and Zn).
All enzymes that contain tightly bound metal ions are termed metalloenzymes .
Properties of enzymes :
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 a 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).
Properties of enzymes :
E. Regulation
Enzyme activity can be regulated, that is, enzymes can be activated or
inhibited, so that the rate of product formation responds to the needs of the
cell.
F. Location within the cell
Many enzymes are localized in specific organelles within the cell. Such
compartmentalization serves to isolate the reaction substrate or product from
other competing reactions. This provides a favorable environment for the
reaction, and organizes the thousands of enzymes present in the cell into
purposeful pathways.
Many Coenzymes, Cofactors & Prosthetic Groups Are Derivatives of B
Vitamins
• The water-soluble B vitamins supply important components of numerous
coenzymes.
Nicotinamide is a component of the redox coenzymes NAD and NADP.
riboflavin is a component of the redox coenzymes FMN and FAD.
Pantothenic acid is a component of the acyl group carrier coenzyme A.
thiamin participates in decarboxylation of -keto acids.
folic acid and cobamide coenzymes function in one-carbon metabolism.
• Structure
• Enzymes are generally globular proteins, acting alone or in larger complexes .
The sequence of the amino acids specifies the structure which in turn determines
the catalytic activity of the enzyme. Although structure determines function.
Enzyme structures unfold (denature) when heated or exposed to chemical
denaturants and this disruption to the structure typically causes a loss of activity.
• Enzymes are usually much larger than their substrates. Sizes range from just 62 amino
acid residues, for the monomer to over 2,500 residues in the animal fatty acid synthase.
• Only a small portion of their structure (around 2–4 amino acids) is directly involved in
catalysis: the catalytic site. This catalytic site is located next to one or more binding sites
where residues orient the substrates.
• The catalytic site and binding site together comprise the enzyme's active site. The
remaining majority of the enzyme structure serves to maintain the precise orientation
and dynamics of the active site.
• the active site is much more than simply a recognition
site for binding substrates. It provides a threedimensional environment that both shields substrates
from solvent and facilitates catalysis. It also binds any
cofactors and prosthetic groups that may be required
for catalysis.
Organisation of enzyme structure (ex lysozyme ). Binding sites in
blue, catalytic site in red and peptidoglycan substrate in black.
• Enzyme structures may also contain allosteric sites where the binding of a small
molecule causes a conformational changes that increases or decreases activity.
• Mechanism:
• Substrate binding
• Enzymes must bind their substrates before they can catalyse any chemical
reaction. Enzymes are usually very specific as to what substrates they bind and
then the chemical reaction catalysed. Specificity is achieved by binding pockets
with complementary shape, charge and hydrophilic / hydrophobic characteristics
to the substrates. Enzymes can therefore distinguish between very similar
substrate molecules .
• Some of the enzymes showing the highest specificity and accuracy are involved in
the copying and expression of the genome. Some of these enzymes have
“proofreading " mechanisms. Here, an enzyme such as DNA polymerase catalyzes
a reaction in a first step and then checks that the product is correct in a second
step.
lock & key model
• The observation that the presence of substrates renders enzymes more resistant to the
denaturing effects of elevated temperatures led Emil Fischer 1894 to propose that
enzymes and their substrates interact to form an enzyme-substrate (ES) complex whose
thermal stability was greater than that of the enzyme itself.
• This early model explains enzyme specificity, but fails to explain the stabilization of the
transition state that enzymes achieve.
• In the lock-and-key model of enzyme action:
 This enzymatic "lock" is referred to as the active site. the active site has a rigid shape.
only substrates with the matching shape can fit . The substrate molecules are aligned in
close proximity and optimal orientation to the functional groups of peptidyl aminoacyl
residues, cofactors, and prosthetic groups responsible for catalyzing their chemical
transformation into products
 the substrate is a key that fits the lock of the active site
Induced fit model:
• In 1958, Daniel Koshland suggested a modification to the lock
and key model: since enzymes are rather flexible structures, the
active site is continuously reshaped by interactions with the
substrate as the substrate interacts with the enzyme.
In some cases, such as glycosidases, the substrate molecule also
changes shape slightly as it enters the active site. The active site
continues to change until the substrate is completely bound, at
which point the final shape and charge distribution is determined.
Catalysis: The mechanism of enzyme action can be viewed from two
different perspectives.
Energy changes occurring during the reaction
Virtually all chemical reactions have an energy barrier (free
energy of activation )separating the reactants and the products.
(Figure 5.4) shows the changes in energy during the conversion of
a molecule of reactant A to product B as it proceeds through the
transition state (high-energy intermediate T*):
1. Free energy of activation: is the difference in free energy
between the reactant and T*, where the high-energy
intermediate is formed during the conversion of reactant to
product. Because of the high free energy of activation, the rates
of uncatalyzed chemical reactions are often slow.
2. Rate of reaction: For molecules to react, they must contain
sufficient energy to overcome the energy barrier of the transition
state. In the absence of an enzyme, only a small proportion of a
population of molecules may possess enough energy to achieve
the transition state between reactant and product.
In general, the lower the free energy of activation, the more molecules have sufficient energy to
pass through the transition state, and, thus, the faster the rate of the reaction.
Dynamics Enzymes are not rigid, static structures; instead they have complex internal dynamic
motions – that is, movements of parts of the enzyme's structure such as individual amino acid
residues, groups of residues forming a protein loop or unit of secondary structure,
, or even an entire protein domain .
• Thermodynamics
• In the presence of an enzyme, the reaction runs in the same direction as it would without the
enzyme, just more quickly.
• Enzymes increase reaction rates by lowering the energy of the transition state.
• First, binding forms a low energy enzyme-substrate complex (ES).
• Secondly the enzyme stabilises the transition state such that it requires less energy to achieve
compared to the uncatalyzed reaction (ES‡).
• Finally the enzyme-product complex (EP) dissociates to release the products.
Enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can
be used to "drive" a thermodynamically unfavourable one so that the combined energy of the
products is lower than the substrates.
THE ANALYSIS OF CERTAIN ENZYMES AIDS DIAGNOSIS
Many enzymes are functional constituents of blood. Examples include pseudocholinesterase,
lipoprotein lipase
 Other enzymes are released into plasma following cell death or injury.
 Assays of enzyme activity typically employ standard kinetic assays of initial reaction rates.
radioimmunoassays (RIA) provide quantitation of the absolute amount of an enzyme or of non
catalytic protein.
Enzymes Assist Diagnosis of Myocardial Infarction
Detection of an enzyme must be possible within a few hours of an MI to confirm a preliminary
diagnosis and permit initiation of appropriate therapy.
The first enzymes used to diagnose MI were aspartate aminotransferase (AST), alanine
aminotransferase (ALT), and lactate dehydrogenase. AST and ALT proved less than ideal,
however, as they appear in plasma relatively slowly and are not specific to heart muscle.
LDH also is released relatively slowly into plasma . Lactate dehydrogenase (LDH) is a
tetrameric enzyme consisting of two monomer types: H (for heart) and M (for muscle) that
combine to yield five LDH isozymes: HHHH (I1 ), HHHM (I2 ), HHMM (I3 ), HMMM (I4 ), and
MMMM (I5). Isozyme I1 predominates in heart tissue, and isozyme I5 in liver.
Today, LDH has been superseded as a marker for MI by other proteins that appear more rapidly in
plasma.
Creatine kinase (CK) has three isozymes: CK-MM (skeletal muscle), CK-BB (brain), and CKMB (heart and skeletal muscle). CK-MB has a useful diagnostic window. It appears within 4–6
h of an MI, peaks at 24 h, and returns to baseline by 48–72 h.
Today, in most clinical laboratories CK has been supplemented by troponin .
Troponin is a complex of three proteins involved in muscle contraction in skeletal and cardiac
muscle but not in smooth muscle. Immunological measurement of plasma levels of cardiac
troponins I and T provide sensitive and specific indicators of damage to heart muscle. Troponin
levels rise 2–6 h after an MI and remain elevated for 4–10 days. In addition to MI, other heart
muscle damage also elevates serum troponin levels . Cardiac troponins thus serves as a marker
of all heart muscle damage.
Enzymes also can be employed in the clinical laboratory as tools for determining the
concentration of critical metabolites. glucose oxidase to measure plasma glucose concentration.
Enzymes are employed with increasing frequency as tools for the treatment of injury and
disease. Tissue plasminogen activator (tPA) or streptokinase is used in the treatment of acute
MI, while trypsin has been used the treatment of cystic fibrosis