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1. Introduction
2. Classification of enzymes
3. Properties:
3.1. Kinetics: Michaelis-Menton &Lineweaver-Burk Plots
3.2. Inhibition: Competitive, Uncompetitive & Non-competitive
3.3. Catalysis: Proximity, electrostatic, acid-base, covalent
4. Cofactors and coenzymes
5. Temperature & pH effects on reactions
6. Application in Industry and medicine
How enzymes work ?
1. recognize very specific substrates,
2. perform specific chemical reactions high
Enzymes make and break specific chemical
bonds of the substrates at a faster rate without
being consumed in the process.
At the end of each catalytic cycle, the enzyme is
free to begin again with a new substrate molecule
• As early as the late 1700s and early 1800s, the digestion of meat by stomach secretions and the conversion
of starch to sugars by plant extracts and saliva were known.
• However, the mechanism by which this occurred had not been identified.
• In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to
the conclusion that this fermentation was catalyzed by a vital force contained within the yeast cells called
"ferments", which were thought to function only within living organisms.
• He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells,
not with the death or putrefaction of the cells.“
• In 1878 German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme, which comes from
Greek ἔνζυμον "in leaven", to describe this process.
• The word enzyme was used later to refer to nonliving substances such as pepsin, and the
word ferment used to refer to chemical activity produced by living organisms.
• In 1897 Eduard Buchner began to study the ability of yeast extracts that lacked any
living yeast cells to ferment sugar.
• In a series of experiments at the University of Berlin, he found that the
sugar was fermented even when there were no living yeast cells in the
• He named the enzyme that brought about the fermentation of sucrose
• In 1907 he received the Nobel Prize in Chemistry for his biochemical
research and his discovery of cell-free fermentation.
• Following Buchner's example; enzymes are usually named according
to the reaction they carry out.
• Having shown that enzymes could function outside a living
cell, the next step was to determine their biochemical nature.
• Many early workers noted that enzymatic activity was
associated with proteins, but several scientists (such as Nobel
laureate Richard Willstätter) argued that proteins were merely
carriers for the true enzymes and that proteins per se were
incapable of catalysis.
• However, in 1926, James B. Sumner showed that the enzyme
urease was a pure protein and crystallized it; Sumner did
likewise for the enzyme catalase in 1937.
• The conclusion that pure proteins can be enzymes was
definitively proved by Northrop and Stanley, who worked on
the digestive enzymes pepsin (1930), trypsin and
• This discovery that enzymes could be crystallized eventually allowed
their structures to be solved by x-ray crystallography.
• This was first done for lysozyme, an enzyme found in tears, saliva and
egg whites that digests the coating of some bacteria; the structure was
solved by a group led by David Chilton Phillips and published in 1965.
• This high-resolution structure of lysozyme marked the beginning of
the field of structural biology and the effort to understand how
enzymes work at an atomic level of detail.
• Enzymes are generally globular proteins, some
are simple proteins and others are conjugated
• Enzymes catalyze reactions without bring
destroyed or chemically changed.
• Enzymes are usually very specific as to which
reactions they catalyze and the substrates that
are involved in these reactions.
• Example: maltase only catalyses the hydrolysis
of maltose and lipase catalyses the hydrolysis of
Properties of enzymes
• A catalyst enhances the rate of reaction but is
not permanently altered.
• Catalysts work by decreasing the activation
energy for a reaction by changing its pathway.
• The structure of the active site of the enzyme
(shape and charge distribution) is used to
optimally orient the substrate for reaction.
• The energy of the enzyme-substrate complex is
then closer to the Transition Stage (Fig 6.1,
p.162 of text)
• An enzyme lowers the activation energy
but it does not change the standard free
energy change (DG) for the reaction nor
the Keq.
• Most enzymes are temperature/pH
sensitive and will not work outside their
normal temperature/pH range because
the enzyme is denatured.
• The correct substrate fits into the
active site of the enzyme like a key
into a lock. Only the right key fits.
• The active site is a specialized region
of the protein where the enzyme
interacts with the substrate.
Lock and Key
Induced-fit Hypothesis
1. Oxidoreductases catalyze redox
reactions. Eg. Reductases or
2. Transferases transfer a group from one
molecule to another. Eg. Transaminases,
3. Hydrolases cleave bonds by adding
water. Eg. Phosphatases or peptidases
• 4. Lyases catalyze removal of groups to
form double bonds or the reverse. Eg.
decarboxylasaes or synthases
• 5. Isomerases catalyze intramolecular
rearrangements. Eg. epimerases or
• 6. Ligases bond two molecules together.
Many are called synthetases. Eg.
• Enzyme kinetics is the quantitative study
of enzyme catalysis.
• Kinetic studies measure reaction rates and
the affinity of enzymes for substrates and
• Kinetics provides insight into reaction
Km is a constant that is characteristic of the enzyme and the substrate . The
lower the value of Km, the greater the affinity of the enzyme for ES complex
When [S] << Km
the velocity will be given by v = (Vmax/Km)[S]. The velocity depends linearly
on [S]. Doubling [S] doubles the rate.
When [S] >> Km,
The equation reduces to v = Vmax, the velocity approaches Vmax , and the
dependence of velocity on substrate concentration approaches a horizontal line
If [S] = Km, the velocity will be one-half of Vmax.
When there is no substrate present ([S] = 0), there is no velocity
Competitive inhibitors bind reversibly to
free enzyme, not the ES complex, to form
an enzyme-inhibitor (El) complex.
At high substrate concentration,
the effect of a competitive
inhibitor can be overcome
Lowering the k1, thus increasing
the Km
uncompetitive inhibition, the inhibitor
binds only to the enzyme-substrate
complex, and not the free enzyme:
enzyme-catalyzed reactions an
inhibitor can bind to both the enzyme and
the enzyme-substrate complex:
A noncompetitive inhibitor binds at a site other than the active site of the
enzyme and decreases its catalytic rate by causing a conformational change in
the three-dimensional shape of the enzyme. The effect of a noncompetitive
inhibitor cannot be overcome at high substrate concentrations.
Effect : No change in Km
• irreversible inhibitors form a reversible non-covalent complex with
the enzyme (EI or ESI) and this then reacts to produce the
covalently modified EI* called inactivation rate.
• EI may compete with ES, binding of irreversible inhibitors can be
prevented by competition either with substrate or with a second,
reversible inhibitor
- Kinetic study is not enough to study how
enzyme catalyze biochemical reactions.
- Need other way : catalysis.
- Factors contribute to enzyme catalysis :
proximity and strain effects
electrostatics effects
acid-base catalysis
covalent catalysis
Factors influencing catalysis
(1) Proximity and Strain Effects
• Substrate must closely approach the
catalytic site with proper orientation.
• The more tightly the active site can bind
the substrate while it is in its transition
state, the greater the rate of the reaction.
Factors influence catalysis…….
(2) Electrostatic Effects
• the strength of electrostatic interactions is related to the
capacity of surrounding solvent molecules to reduce the
attractive forces between chemical groups.
• the dielectric constant near the active site is often low,
this may influence the chemical reactivity of the
• weak electrostatic interactions in both the active site and
the substrate, are believed to contribute to the catalysis.
Factors influence catalysis
(3)Acid-Base catalysis
• Chemical groups can often be made more
reactive by adding or removing a proton.
• Enzyme active sites contain side chain
groups that act as proton donors or
In covalent catalysis, a transient covalent bond is formed between the
enzyme and the substrate.
Consider the hydrolysis of a bond between groups A and B:
In the presence of a covalent catalyst (an enzyme with a nucleophilic
group X:) the reaction becomes
The higher the temperature, the higher the reaction rate.
The rates of enzyme-catalyzed reactions also increase with
increasing temperature.
Changes in [H] ion can affect the ionization of active site
If a substrate contains an ionizable group, a change in pH
may alter its capacity to bind to the active site.
Changes in ionizable groups may change the tertiary
structure of the enzyme.
REGULATION- refer from textbook
- to maintain an orderly state
- consevation of energy
- responsivenes to envirinmental change
- genetic control
- covalent modification
- allosteric regulation
- compartment
Cofactors & Coenzymes
• Many enzymes require the presence of small,
nonprotein units or cofactors to carry out their
particular reaction.
• Cofactors can be :
– inorganic ions, exp: Fe2+ and Cu2+ and the alkali
and alkaline earth metals (e.g., Na+, K+, Mg2+, and
– a complex organic molecule called a coenzyme.
Exp :
• Nicotinamide adenine dinucleotide (NAD)
• Nicotinamide adeninedinucleotide phosphate (NADP)
• Flavin adenine dinucleotide (FAD)
• flavin mononucleotide (FMN)
Enzyme Application : In Industry
Some examples :
1. In Textile industry : Amylase enzyme as a sizing agent
2. In Sugar industry : Amylase to reduce the starch content of sugar cane syrup
3. In energy related Ind : Lipase to speed up the esterification process of
4. In Beverages : Pectinases for juice clarification
5. In Detergent ind : amylase & protease to enhance the cleaning power of
6. Enzyme cellulase breaks down cellulose to beta-glucose in the ruminating
chambers of herbivores; it is also used in processing coffee and fermentation of
biomass into biofuels; it is a fading agent in textile industry.
Enzyme technology currently plays a role in two aspects of medical practice:
diagnosis and therapy.
A. Diagnosis :
1. Enzyme activity as an indicator of diseases.
Exp : blood levels of acid phosphatase (enzyme) for diagnosing prostatic
carcinoma (a urinary tract tumor that occurs in males).
2. Enzymes as reagents.
Exp : enzyme urate oxidase is used to measure blood levels of uric acid (which is
usually high in patients with gout. )
B. Therapeutic
The use of enzymes in medical therapy has been limited because when
administered to patients, enzymes are often rapidly inactivated or degraded.
Exp : The enzyme asparaginase is used to treat of cancer. It catalyzes the
conversion of Asparagine to Aspartate .Infusing asparaginase reduces the
blood's concentration of asparagine and often causes tumor regression.
Antioxidant co-enzymes
Antioxidant co-enzymes:
– glutathione peroxidase
– superoxide dismutase (SOD),
works with catalase, scavenge and
neutralize cell-damaging free radicals
by turning them into stable oxygen and
H2O2, and then into oxygen and water.
A process that dissolves blood clots
• Enzymes
– Trypsin
– chymotrypsin
• One use of enzymes, is in the case of
thrombosis, this is when blot clots form in
damaged blood vessels, if these clots are
carried to an small artery and may become
blocked a heart attack or stroke can be
Sports injuries
• Sports injuries are often treated with protease
enzymes because of their ability to reduce
inflammation and speed the healing of bruises,
swelling, and other injuries
• A study conducted by Dr M.W. Kliene and his
coworkers at the Sports Medicine Investigation
Center in Grunwald, Germany researched the
effectiveness of proteolytic enzymes on 100
• The results favored the enzyme treated subjects.
Seventy-six percent evaluated the success rate
as ‘good’ with protease enzymes.
Several studies showed that when enzymes
were taken before surgery, the swelling from
the injury left around seven days quicker, on
average, than those taking a placebo; post
surgery recovery was also much more rapid.
Digestive aid
• A concentrated form of pancreatic amylase
was available as Diastase Vera.
• Amylase obtained from malt—produced
from the action of Aspergillus oryzae on
sterilized rice hulls or on wheat bran—was
also marketed,
• Diastase was mainly indicated for use by
people who had overindulged in starchy
food such as pasta
1. Enzymes are biological catalysts which enhance reaction rates. Enzymes are
specific to the types of reactions they catalyze.
2. There are six major enzyme categories: oxidoreductases, transferases,
hydrolases, lyases, isomerases, and ligases.
3. Enzyme kinetics is the quantitative study of enzyme catalysis. Ref : MichaelisMenten, Km and Vmax
4. The most common types of reversible inhibition are competitive, uncompetitive,
and noncompetitive.
5. Allosteric enzymes are enzymes containing multisubunit proteins. The kinetic
properties of allosteric enzymes are not explained by the Michaelis-Menten
6. Several factors contribute to enzyme catalysis: proximity and strain effects,
electrostatic effects, acid-base catalysis, and covalent catalysis.
7. Enzymes are sensitive to environmental factors such as temperature and pH.
Each enzyme has an optimum temperature and an optimum pH.