ENZYMOLOGY

Dian Mulawarmanti
SCHOOL OF MEDICINE
Hang Tuah UniversitY
2006
Standard Protein Structure

 .
Amino Acid Sequence

3-D Structure

Protein Function

ENZYME
Struktur primer
 Urutan asam amino pada rantai peptida
dari ujung amino bebas (awal) sampai
ujung karboksil bebas (akhir)

 Dihubungkan ikatan peptida

 Protein
Chemistry

Figure 3-2. The structural components of a protein. A

protein consists of a polypeptide backbone with attached
side chains. Each type of protein differs in its sequence
and number of amino acids; therefore, it is the sequence of
the chemically different side chains that makes each
protein distinct. The two ends of a polypeptide chain are
chemically different: the end carrying the free amino group
(NH3+, also written NH2) is the amino terminus, or N-
terminus, and that carrying the free carboxyl group (COO ,
also written COOH) is the carboxyl terminus or C-terminus.
The amino acid sequence of a protein is always presented
in the N-to-C direction, reading from left to right
STRUKTUR SEKUNDER PROTEIN
ß- pleated sheet
random coil

 .
 STRUKTUR TERTIER PROTEIN
1 polipeptida  monomer
ada celah
ikatan hidrogen, gaya van der waals
 .
STRUKTUR KWARTER PROTEIN
bentuk dimer, tetramer, oligomer

 .
How Protein Folds
ENZYMES : GENERAL PROPERTIES
Introduction to Enzymes

 Enzymes
biological catalysts  maintain animal
homeostasis.

 Because of their role in maintaining life

processes, the assay and pharmacological
regulation of enzymes have become key
elements in clinical diagnosis and therapeutics
Enzyme History
 1833 Payen & Peroz - alcohol precipitate of barley
holds heat labile components (proteins) that converts
starch to sugars
 1878 Kuhn - coins term 'enzyme' : Greek "in yeast"

 1897 Hans & Eduard Buchner - yeast

'juice' + sugars (jelly) = bubbled gas & alcohol

 1898 Ducleaux - uses suffix "ASE" for enzyme names

 1900 E. Fischer - stereospecificty of enzymes is
discovered
ENZYMES are

 Biological catalysts and enhance reaction

rates
 Most are proteins except Ribozymes
 Have primary, secondary, tertiary and
quaternary structures
 Contain an active site where a substrate
binds and is converted to products
 One polypeptide chain has one active site
 Show Stereochemical and structural
specificity
 The components :

Protein, except for a class of RNA

modifying catalysts known as
ribozymes. Ribozymes are molecules of
ribonucleic acid that catalyze reactions
on the phosphodiester bond of other
RNAs
Enzymes
tissues and fluids of the body.

 Intracellular enzymes :
metabolic pathways.

 Plasma membrane enzymes :

response to extracellular signals

 Enzymes of the circulatory system :

for regulating the clotting of blood.
 Eukaryotic Cell

Figure 1-31. The major features of eucaryotic cells. The drawing depicts a typical animal cell, but almost all the same components are found in
plants and fungi and in single-celled eucaryotes such as yeasts and protozoa. Plant cells contain chloroplasts in addition to the components shown here,
and their plasma membrane is surrounded by a tough external wall formed of cellulose
Enzyme characteristics
 Proteins
 Biological catalysts
 Very efficient catalysts
 Very specific catalysts
 Reduce energy of activation for reaction (by
binding the transition state)
 Carry out catalysis in a special region of the
molecule, the Active-site
 Exhibit special kinetics
 Subject to regulatory control of various sorts
ENZYME ACTIVE SITE
Active site includes side chains from different
regions of the enzyme
E + S <---> [ES] <---> E + P
Enzymes are true catalysts

 enhance the rate of a specific rxns, but

do not participate in the rxn
 do not shift the equilibrium of reactions
 are regenerated following the reaction
(therefore, not consumed in reaction)
 effective in minute concentrations
compared to the [S]
Catalytic efficiency
 A typical enzyme will convert around a
thousand molecules of substrate to
product in 1 second! Some will
convert as many as a million!! There are
of course slower enzymes as well
Substrate Specificity -- Highly specific for
the reactants, or substrates with degree of
substrate specificity dependent on enzyme
Most enzymes are highly specific for their
particular S (one E -- one S)
 Some enzymes will only catalyze one type
of reaction for one type of compound
 In some cases for only one substrate and thus
very high and selective specificity
 Some enzymes recognize structurally
similar substrates
 Thus they have broad specificity
 thrombin Glucose oxidase

Type of reaction hydrolisis oxidation

catalyzed

Type of bond peptide Sugar hemiacetal

Structure about bond between Arg and Gly glucose

Stereospecificity both must be L amino acids D configuration

Gugus Fungsional
 = gugus reaktif
 Letak pada gugus rantai
peptida
 Yang dapat bereaksi hanya
pada permukaan protein
 Menentukan sifat-sifat umum
protein
Mechanism of Enzyme Action

 E + S <---> [ES] <---> E + P

 enzymes catalyze reactions by lowering

the energy of activation... Ea
ACTIVATION ENERGY CONCEPT
EA : energi yg diperlukan karena
hambatan E (energi barier)

= jumlah energi yg diperlukan utk

membawa semua molekul dalam 1
mol bahan pada suhu ttt dari keadaan
awal ke keadaan transisi

∆ G = perubahan energi bebas

= selisih energi awal dan akhir, tidak
dipengaruhi adanya katalisator
Catalase:

2H2O2 ---> 2 H2O + O2

condition Ea(cal/mol) Rate
(lt/mol/sec)
no catalyst 18,000 1.0 x 10-7
Fe catalyst 10,000 56.0
catalase 2,000 4.0 x 106
Enzyme Classifications
IUBMB International Union Biochemistry & Molecular Biology

4 digit Numbering System [1.2.3.4.]

 1st #... one of 6 Major Classes of Enzyme
Activity
 2nd #... a subclass (type of bond acted upon)
 3rd #... a subclass (group acted upon, cofactor
required, etc...)
 4th #... a serial number… (order in which
enzyme was added to list)
The 'ENZYME' data bank contains the following data for
each type of characterized enzyme for which an EC
number has been provided:

 - EC number
 - Recommended name
 - Alternative names (if any)
 - Catalytic activity
 - Cofactors (if any)
The Enzyme Data Bank based on
the following:

 Search by EC number
 Search by enzyme class
 Search by description or alternative
name
 Search by chemical compound
 Search by cofactor
Enzyme Classifications

1.Oxidoreductases
2.Transferases
3.Hydrolases
4.Lyases
5.Isomerases
6.Ligases
 Enzyme Classifications
IUBMB International Union Biochemistry & Molecular Biology

1.Oxidoreductases:
Act on many chemical groupings to add or
remove hydrogen atoms.
2.Transferases:
Transfer functional groups between donor and
acceptor molecules. Kinases are specialized
transferases that regulate metabolism by
transferring phosphate from ATP to other
molecules.
3.Hydrolases:
Add water across a bond, hydrolyzing it.
 Enzyme Classifications
IUBMB International Union Biochemistry & Molecular Biology

4.Lyases:
Add water, ammonia or carbon dioxide across
double bonds, or remove these elements to
produce double bonds.
5.Isomerases:
Carry out many kinds of isomerization: L to D
isomerizations, mutase reactions (shifts of
chemical groups) and others
6.Ligases:
Catalyze reactions in which two chemical groups
are joined (or ligated) with the use of energy
from ATP.
EC 3.1.1.7. is acetylcholine esterase:

 3: Hydrolase, so adds water to molecules.

 1: Esterase, so acts on the organic salts of
acids.
 1: Carboxylic acid esterase, so acts on
R-COO-R′ groups.
 7: Acetylcholine esterase.


EC MAJOR CLASSES of Enzymes some common

functional classes of enzymes

1. Oxidoreductases... [ dehydrogenases]
catalyzes oxidation reduction reactions, often using coenzyme as
NAD+/FAD
Alcohol dehydrogenase [EC 1.1.1.1]
ethanol + NAD+ ---> acetaldehyde + NADH

2. Transferases... catalyze the transfer of functional

group
Hexokinase [EC 2.7.1.2] D-glu + ATP ---> D-glu-6-P + ADP

3. Hydrolyases… catalyzes hydrolytic reactions adds water across C-C

bonds
Carboxypeptidase A [EC 3.4.17.1]
[aa-aa] n + H2O ---> [aa-aa] n-1 + aa

EC MAJOR CLASSES of Enzymes some
common functional classes of enzymes

4. Lyases... adds or removes groups to C=C

bonds
Pyruvate decarboxylase [EC 4.1.1.1]
pyruvate ---> acetaldehyde + CO2

5. Isomerases… [mutases] catalyze isomerizations

Maleate isomerase [EC 5.2.1.1]
maleate ---> fumarate

6. Ligases… condensation of 2 substrates with splitting o

ATP
Pyruvate Carboxylase [EC 6.4.1.1]
PYR + CO 2 + ATP ---> OAA + ADP + P
 Holoenzymes : Complex enzymes
 Apoenzyme : the protein component
 the coenzyme or prosthetic group : non-
protein component

 prosthetic group describes a complex in which

the small organic molecule is bound to the
apoenzyme by covalent bonds;

 when the binding between the apoenzyme and

non-protein components is non-covalent, the
small organic molecule is called a coenzyme
Cofactors

 Most enzymes use their amino-acid side chains to

bind and manipulate substrates, like fingers;
however, some use additional tools, called cofactors.
These may be further subdivided into:

 Coenzymes, which are soluble and may diffuse

between different enzymes. ATP, NADH.
 Prosthetic groups, which are strongly bound to the
enzyme, either covalently, or by other means. Haem,
FeS clusters.
 Metal ions, which are often bound by dipole
interactions with histidine and other amino acids with
lone-pairs. Fe2+ in catalase.
 Role of Coenzymes

 The functional role of coenzymes is to act as

transporters of chemical groups from one
reactant to another.

 The chemical groups carried can be as simple as

the hydride ion (H+ + 2e-) carried by NAD or
the mole of hydrogen carried by FAD; or they
can be even more complex than the amine (-
NH2) carried by pyridoxal phosphate.
Two very important coenzymes are ATP
and NADH

 ATP, ADP and AMP

carry energy using 'high energy' phosphate
bonds, derived from nucleic acids in food.

 NAD(P)H and NAD(P)

reducing power as hydride ions (H−).
derived from vitamin B3 (niacin/nicotinic acid)
 Many prosthetic groups and coenzymes
are water-soluble derivatives of
vitamins.

 dietary vitamin ↓  malfunction of

enzymes, which lack sufficient cofactors
derived from vitamins to maintain
homeostasis.
 Many are derived from vitamins

Coenzyme . Vitamin Precursor

Coenzyme A Pantothenic acid

Thiamine pyrophosphate Thiamine (B1)

Tetrahydrofolate Folic acid

NAD, NADP Niacin

FAD, FMN Riboflavin (B2)

Prosthetic groups
 A example is haem, which is found in cytochromes
and haemoglobin, and carries oxygen and/or
electrons.

Haem
Prosthetic groups
 Iron-sulfur clusters also carry electrons, and are
found in ferredoxin, an important intermediate in
photosynthesis

Iron-sulfur clusters
METAL ION CATALYSIS
Fe, Zn, Mn
THE DIFFERENCES BETWEEN
CATALISATOR IN ORGANIK AND
ENZYME

 CATALISATOR  ENZYME
INORGANIK
1. Protein 
1. H+, OH-,Pt biocatalisator
2. E aktivasi ↓ 2. E akt ↓ ↓
3. Specific reaction
4. Heat labile
Enzyme Relative to Substrate
Type
 For example,
succinic dehydrogenase (SDH) always catalyzes an
oxidation-reduction reaction and its substrate is invariably
succinic acid,
 alcohol dehydrogenase (ADH) always catalyzes oxidation-
reduction reactions but attacks a number of different alcohols,
ranging from methanol to butanol. Generally, enzymes having
broad substrate specificity are most active against one particular
substrate.
 In the case of ADH, ethanol is the preferred substrate.
 : Definitions of Enzyme Activity:
 a physical properties of an enzyme... most often measured by
relative rate that substrate ---> product

 1 unit ACTIVITY that amount enzyme protein which converts

 1 umole substrate per min at 25oC & optimal pH

 1 unit SPECIFIC ACTIVITY

# units per mg of protein present
(e.g., 37umole /min/mg protein)

 1 unit MOLECULAR ACTIVITY

# units per umole of purified enzyme
(e.g., 12 units/umole enzyme)
Enzymes can bind substrates by any sort of
chemical bond. Different sorts of bonding may
lead to different sorts of catalysis.

 Ionic interactions.
 Hydrophobic interactions.
 Covalent interactions.
 Hydrogen bonding.
 Dipole interactions
Zymogen
proenzyme
 SECRETION IN ACTIVE

 TO PROTECT FROM AUTODIGESTION

 FOR ACTIVATION ARE NEEDED :

PROTEOLITIC ENZYMES / H+
 CHANGE CONFORMATION
 CATALYTIC SITE OPEN
 ISOZYME

 CATALYZES THE SAME REACTION

 CHEMICAL, PHYSICAL, IMUNNE
CHARACTERISTIC  << DIFFERENT
 LOCATE VARIATION IN TISSUE CAN
DIAGNOSIS DISEASE
 THE DETECTION of specific LDH isozymes in
THE BLOOD is HIGHLY diagnostic of TISSUE
DAMAGE, such as occurs during cardiac
infarct.
 isozyme
 lactate dehydrogenase (LDH)

 LDH is :
¤ a tetrameric enzyme composed of two
different protein subunits; the subunits are
known as H (for heart) and M (for skeletal
muscle).

¤ These subunits combine in various

combinations  5 distinct isozymes
isozymes

 H isozyme ~ heart tissue

 M isozyme ~ skeletal muscle and liver
 These isozymes all catalyze the same
chemical reaction, but they exhibit
differing degrees of efficiency
 Isozymes :I1=H4, I2=H3M , I3=H2 M2,
I4=HM3, I5=M4
isozyme
curve
Enzyme catalysis rates are affected
four important factors

 Temperature.
 pH.
 Substrate concentration.
 Activator/inhibitor
Acetylcholine (ACh) + water + esterase
→ acetic acid + choline + esterase

At a given pH and temperature, the rate of

hydrolysis of ACh depends on :
 (substrate) (ACh).

 (substrate) (water).

 (enzyme)

 (ES) complex
. ANY QUESTIONS ??