Yrd. Doç. Dr. Bekir Engin Eser
Zirve University
EBN School of Medicine
Department of Medical Biochemistry

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One of the most important function of proteins is their ability to catalyze biological reactions
This group of proteins are called enzymes
In addition to proteins, there are also some enzymes which are in nucleic acid structure (RNA) http://cnx.org/contents/585c4d6a-0aca-465c-97d0-68272d2bb614@1.5:15
Introduction to Anatomy & Physiology Stelios Kolomvounis

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Catalytically active biological molecules: mostly proteins, also some RNA molecules
Accelerate reaction rates (up to 10 7 rate enhancement)
Reaction specificity towards certain substrates
Milder reaction conditions
Capacity for regulation
Use metal or organic molecule cofactors to assist their functioning
Free
Energy
Reactants http://en.wikipedia.org/wiki/File:GLO1_Homo_sapiens_small_fast.gif
Activation energy
Products
Progress of the reaction

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Biological catalysts rate constant enzyme
(&cofactor
/coenzyme) substrate
Enzyme-substrate complex product
ü   Enzymes carry out transformations on substrates at
physiological conditions
ü   Enzymes are selective; they only work on certain substrates

1) Catalytic Effect: Rate enhancement of the reactions up to 10 7 times
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Enzymes generally have a rate of ~2-1000 s -1
(turnover number)
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“ Turnover number ” is the number of product molecules that is produced by the enzyme in a defined period of time (s or min)

2) Active Site: Enzymes have an active region on their surface (a pocket), called “ active site ”
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Active site pocket has a certain shape that will fit to the substrate molecule
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Enzymes discriminate their substrates by the binding ability of their active sites only to certain substrates – specificity of enzymes

1) Lock and key model (anahtar kilit modeli) rigid active site
2) Induced Fit (indüklenmi ş uyma) model
Flexible active site http://wps.pearsoned.com.au/sf2_2/134/34366/8797829.cw/content/index.html
http://biochemistryquestions.wordpress.com/2008/07/15/induced-fit-model-of-enzyme-substrate-interaction/

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Catalysis (chemical transformations) takes place at the active site
Substrates, cofactors and coenzymes bind to the active site
Amino acid residues that carry out catalysis are found at the active site (catalytic residues)
Within the active site, substrates are brought into close proximity with the other substrates and/or cofactors & coenzymes (so that the chemical reaction can take place)
Active site can also protect substrates from water and from the properties of the surrounding environment, creating a local environment (polarity, hydrophobicity, acidity etc.)

3) Specificity
(Selectivity):
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Each enzyme is specific for a certain single substrate or for a small group of similar substrates
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Each enzymes in general catalyzes only one type of reaction
Specificity can be 1) at the level of binding to the active site 2) at the level of reaction (inhibitor) http://droualb.faculty.mjc.edu/Course%20Materials/Physiology%20101/Chapter%20Notes/Fall%202011/chapter_3%20Fall%202011.htm

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Enzymes are also stereospecific catalysts
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They typically catalyze reaction of only one stereoisomer, but not the other (D or L)
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Enzymes can also convert nonchiral substances to chiral products http://nptel.ac.in/courses/104103018/module3/lec6/images/11.png

achiral

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4 ) Cofactors and coenzymes:
Many enzymes require cofactors or coenzymes for their activity
Cofactors and coenzymes might be bound to the active site or other parts of the enzyme
Cofactors: metal ions (iron, copper, zinc)
Tightly bound to the active site-prosthetic group
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Coenzymes: complex organic molecules
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Many vitamins and vitamin derivatives are coenzymes
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Some of the coenzymes are tightly bound to the active site (prosthetic groups)
Some of them binds to the active site only during the catalysis
Apo enzyme – no cofactor/coenzyme bound
Holo enzyme – cofactor/coenzyme bound https://adapaproject.org/bbk/tiki-index.php?page=Leaf%3A+How+do+proteins+and+RNAs+control+cellular+chemical+reactions%3F

Cofactors: Fe 2+ , Fe 3+ , Mg 2+ , Zn 2+ , heme group (iron + organic group)
Coenzymes: Flavin mononucleotide (FMN), Nikotin adenin dinucleotide (NAD), thiamin (vitamin B1), pyridoxal phosphate (active form of Vitamin B6), heme group http://lecturer.ukdw.ac.id/dhira/Metabolism/Enzymes.html

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5) Regulation : The activity of enzymes are regulated
Enzyme regulation can be in many ways:
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1) By small molecules that bind to the parts of
—   the enzyme other than the active (allosteric control)
2) By inhibitors that bind in the place of the substrate at the active site (feedback inhibition)
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3) By covalent modifications on some of the amino acids of the enzyme (serine phosphorylation)
4) By regulation of enzyme synthesis at gene expression level (induction and repression of protein synthesis)

1) Oxido-Reductases (redox)
2) Transferases
3) Hydrolases
4) Lyases
5) Isomerases
6) Ligases
Enzymes are classified by their reaction type

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Enzymes that catalyze oxidation and reduction
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These enzymes oxidize or reduce their substrates by transferring electron, hydrogen or oxygen
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In general oxidoreductases use coenzymes such as
NADH, FADH
2
, FMN as electron/hydrogen donors or acceptors in their reactions http://quizlet.com/16563961/biochem-enzymes-l1-properties-flash-cards/

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Enzymes that catalyze transfer of groups such as phosphoryl, glycosyl or methyl are called transferases http://quizlet.com/16563961/biochem-enzymes-l1-properties-flash-cards/

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Enzymes that catalyze hydrolytic cleavage
(reaction with water) of covalent bonds (such as C-C, C-O, C-N) are called hydrolases
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Catalysis of hydrolysis reaction

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Enzymes that catalyze cleavage of C-C, C-O,
C-N or C-S bonds (by ways other than hydrolysis and oxidation) are called lyases pyruvate decarboxylase https://lookfordiagnosis.com/mesh_info.php?term=pyruvate%20decarboxylase&lang=1

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Enzymes that catalyze structural or geometric changes within a molecule (without changes in the atom content) are called isomerases
Isomers
Stereoisomers
Constitutional
Isomers
Enantiomers Diastereomers

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Enzymes that catalyze bond formation (C-O, C-S, C-N and C-C)
Two molecules are joined together (ligated)
These reactions are
“condensation” type reactions (generally a small molecule is also formed)
Bond formation is generally coupled to the hydrolysis of
ATP

Enzymes catalyze reactions (make reactions faster) by lowering the activation energy of the reaction http://classes.midlandstech.edu/carterp/courses/bio225/chap05/lecture2.htm

In general, enzymes lower the activation energy by carrying out the reaction in multiple steps (through many intermediates) http://en.wikipedia.org/wiki/Catalysis

Homo-octamer
Generally obtained by X-ray diffraction of protein crystals

Phenylalanine hydroxylase (PAH):
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Catalyzes conversion of phenylalanine into tyrosine
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PAH is a member of oxidoreductases
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PAH is a liver enzyme
PAH needs Fe 2+ as cofactor and biopterin as a coenzyme
Its deficiency leads to the disease known as phenylketonuria http://openwetware.org/wiki/IGEM:IMPERIAL/2009/Encapsulation/PKU
Fe 2+

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SOD is an enzyme that functions as an antioxidant
SOD catalyzes conversion (dismutation) of superoxide to oxygen and hydrogen peroxide (H
2
O
2
SOD is a member of oxidoreductases
)
There are many types of SOD depending on their different cofactor requirements (Cu/Zn, Fe, Mn or Ni)
SOD is an important enzyme that is linked to aging http://textbookofbacteriology.net/nutgro_4.html

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Trypsin functions in the digestion of proteins (proteases)
Produced in pancreas
Trypsin cleaves the polypeptide chain after lysine or arginine amino acids
Trypsin is a member of hydrolases (serine protease)
Trypsin does not have any requirement for a cofactor or coenzyme
Trypsin needs to be activated by another enzyme called enterokinase http://www.liv.ac.uk/~agmclen/Medpracs/practical_3/theory_3.html

Catalytic rate , is the amount of product produced or the amount of substrate consumed by a certain amount of enzyme in unit time
Turnover number: (also termed catalytic site in unit time k cat
) is defined as the number of molecules of substrate that an enzyme can convert to product per
1 . Substrate concentration
2 . Temperature – depends on the organism
3 . pH – each enzyme has an optimum working pH range

1.Substrate Concentration:
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As the substrate concentration increases, the rate of the enzyme catalysis increases (at constant enzyme concentration)
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The increase stops at certain substrate concentration; if all the enzyme molecules become saturated with the substrate, there is no more increase of the rate
V
V
MAX
V
O
[ S ]

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In an enzyme catalyzed reaction, the rate of catalysis increase with increasing temperature
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In general, an increase of 10 ºC in temperature leads to doubling of the enzyme activity (at a certain temp. range)
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However, since enzymes are proteins, they start to denature after certain temperature (40-45ºC) and the activity of the enzyme starts to decrease
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Optimum Temperature:
At optimum temperature enzyme has the highest activity
(highest turnover number)

3. pH
The rate of enzymatic reactions are affected by H + ion concentration of the medium
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Optimum pH is the pH at which the activity of the enzyme is maximum b a
4 5 6 7 8 9 10 pH

a) Optimum pH in a small range – bell-shaped curve b) Optimum pH in a wider range – plateau curve
In general, optimum pH range for enzymes are between 5-8
bell-shaped curve curve with a plateau at low pH
Optimum pH
4 6 8 10 12 pH
Trypsin
2 3 4 5 6 7 pepsin pH

Effects of pH changes on the catalytic activity of enzymes
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Very low (acidic) and very high (basic) pH values lead to the denaturation of the enzyme – enzyme loses its activity when it is denatured
Denaturation is sometimes reversible, sometimes irreversible
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The ionization states of the amino and carboxyl groups of amino acids change upon change in pH
Changes of the ionization states of the active site residues affect catalysis (might effect substrate binding or chemical reaction)
-The ionization state of the substrate might also be affected by the pH

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Many enzymes contain small organic molecules and/or metal ions that help their catalysis
Cofactors & coenzymes are required for enzyme function
These groups directly participate in substrate binding or in catalysis
Cofactors: Metal ions
Coenzymes: organic molecules
Prosthetic groups: Cofactors and coenzymes which are tightly bound to the enzyme; they are often attached by covalent bonds

Coenzymes &
Cofactors
Cofactors Coenzymes
Prosthetic groups loose
(reversible) binding
Prosthetic group loose
(reversible) binding

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Almost 35% of all enzymes contain a metal cofactor
Most of the metal cofactors are prosthetic groups and they are tightly bound at the active site of the enzymes
These enzymes are also called as
“ metalloenzymes ”
Metal ions might be bound to the amino acid side chains or they might be complexed to groups such as heme group or iron-sulfur clusters
Fe 2+ , Zn 2+ , Mn 2+ , Ni 2+ are some of the examples

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1) They participate in redox reactions by transfer of electrons
2) They can form covalent bonds with intermediates
3) They act as Lewis acids or bases to make substrates more electrophilic (electron poor) or nucleophilic (electron rich) – make more reactive
4) Facilitate binding and orientation of substrates

sulfur iron
transport of oxygen redox catalysis http://www.pnas.org/content/98/26/14751/F1.large.jpg iron-sulfur clusters redox catalysis

Tyrosine hydroxylase
Fe 2+ coordinated by three amino acids
2 histidine, 1 glutamate redox enzyme – iron is the redox active part of the enzyme

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Many of the coenzymes are vitamins or derived from vitamins (especially B vitamins)
Thiamine pyrophosphate (Vitamin B1), pyridoxal phosphate (Vitamin B6), riboflavin (Vitamin B2), NADH, FADH
2
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FMN are some examples of coenzymes
Coenzymes can be prosthetic groups
(bind enzymes tightly) or they can bind enzymes transiently (reversibly - cosubstrates)
They generally bind to the active site

http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Reference_Tables/Structures_of_common_coenzymes

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Coenzymes serve as recyclable shuttles (they are regenerated and reused)
Main roles:
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1) They can stabilize reactive species (NADH; hydride ion)
2) They help enzymes to transport chemical groups from one molecule to another molecule
(such as methyl, hydride, phosphate, acetyl etc.)
2) They can also serve as adaptors that facilitate binding and recognition of other chemical molecules (e.g. substrates) by their target enzymes

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Discovery of new types of chemical reactions and biological pathways
Enzymes are potential drug targets in pathogenic microorganisms
Deficiency & mutation of enzymes lead to many diseases: identification of the mechanism of an enzyme leads to the development of potential treatments
Many enzymes are useful for synthesis of high-value products such as biofuels and chemicals enzyme
(&cofactor) substrate Enzyme-substrate complex product