Lecture of Enzymes.
Lecture of Enzymes.
Lecture of Enzymes.
Enzymes:
Purification of Enzymes:
The intracellular enzymes should be liberated from the cell by:
1-Grinding the tissue with sand
2-Using homogenizer
3-By rupturing the cell wall by dehydration with acetone and then filtration to dissolve lipid membrane.
4-protein extraction by:1- dialysis 2-suitable buffer 3-Adsorption on different surface active substances 4-
By glycerol.
5-Recently the most important methods are electrophoresis & chromatography.
Q/What are the types of enzymes?
1
A/ 1-digestive enzymes: Secreted by stomach, pancreas, & salivary glands
2-Food enzymes: exist in food
3-Exist in all cells &organs. Act to maintain normal function of cells.
A. Enzymatic activity: _
The catalytic action of an enzyme, its activity, is measured by determining the increase in the
reaction rate under precisely defined conditions.
International unit (IU) for enzymes activity: can be defined as: (The amount of enzymes which,
under given assay conditions, will catalyze the conversion of (l mmol of substrate per minute).
Note: other unit called (katal)= mol /second.
Reaction rates: are expressed as the change in concentration per unit of time (mol. L–1.S–1).
Since the catalytic activity of an enzyme is independent of the volume, the unit used for enzymes is usually
–1
turnover per unit time, expressed in katal (katal= mol S ). However, the international unit U is still more
–1
commonly used (μmol turnover min ; 1 U =16.7 nanokatal).
.
Q/What are the mechanisms for the binding between active site of an enzyme & substrate?
A/There are two main theories for the explanation of binding between active site of an enzyme & substrate:
1-Lock & Key theory:
To fit the interaction between substrate & enzyme, the enzyme has amatching shpe for substrate at
the active site:
Figure: The interaction between substrate & enzyme according to key & lock theory
2-Induced Fit Theory (Hand &Glove):
The active site of the enzyme changing to be complementary to that of the substrate only after the substrate
is bound.
Figure: The interaction between substrate & enzyme according to induced fit theory
In 1993 M. Britt introduced the (Shifting specificity) model for enzyme catalysis. This theory is
a mixture of these theories with modifications.
B. Reaction and substrate specificity: _
The multiplicity of enzymes, their specificity (the ability to discriminate between reactants), and their
susceptibility to regulation give cells the capacity to lower activation barriers selectively.
The action of enzymes is usually very specific. It’s the most significant property
This applies not only to the type of reaction being catalyzed, but also to the nature of the reactants
(―substrates‖) that are involved (substrate specificity).
(Reaction specificity): The ability of an enzyme to catalyze one specific reaction & essentially no others.
(Substrate specificity): A particular enzyme acts only on particular chemical grouping e.g., Alcohol
dehydrogenase acts on alcohol group (C-OH).
(Optical specificity): The complementary of substrate & enzyme must include the attachment at 3
different sites of attachment to distinguish between identical group on substrate
Glycero
l Glycerolkinas
e L3Phosphoglycerate
( procheral ) AT
P (Chiral) ADP
In the Figure B, this is illustrated schematically using a bond-breaking enzyme as an example.
Type A (top): Highly specific enzymes catalyze the cleavage of only one type of bond, and only when the
structure of the substrate is the correct one.
Type B (middle): Other enzymes have narrow reaction specificity, but broad substrate specificity.
Type C: enzymes (with low reaction specificity and low substrate specificity, bottom) are very rare.
C. Classification & Nomenclature of Enzymes:
1. By using the suffex (-ase) after the reactant name. e.g. Urease, Arginase….etc.
2. Arbitrary (traditional) names. e.g. trypsin, & chemotrypsin…etc.
3. Enzymes are classified according to the type of reaction they catalyze (six types). All enzymes have
the Enzyme Catalogue with a four-digit Enzyme Commission number (EC number). The first digit
indicates membership of one of the six major classes. The next two indicate subclasses and
subsubclasses. The last digit indicates where the enzyme belongs in the subsubclass.
For example:-lactate dehydrogenase has the EC number 1.1.1.27(class 1, oxidoreductases; subclass 1.1, CH–
+
OH group as electron donor; sub-subclass 1.1.1, NAD(P) as electron acceptor).
#A system of classification has been developed that takes into account both their:1- reaction specificity
and their 2-substrate specificity.
Enzymes with similar reaction specificities are grouped into each of the six major classes:
(Class 1)=Oxidoreductases catalyze the transfer of reducing equivalents from one redox system to another.
They require coenzymes e.g.,
+ Lactate Dehydrogenase +
Pyruvate + NADH + H Lactate + NAD
OH
O
COO-
- CH3
CH3 H
COO
(Class 2)=Transferases catalyze the transfer of other groups from one molecule to another. Transferases
generally require coenzymes.e.g.,
Hexokinase
Glu cose ATP Glu cose 6 Phosphate ADP
(Class 3)=Hydrolases are also involved in group transfer, but the acceptor is always a water molecule.e.g.,
Ester H 2O Esterase Alcohol Acid
(Class 4)=Lyases, often also referred to as ―synthases‖ catalyze reactions involving either the cleavage or
formation of chemical bonds, with double bonds either arising or disappearing. e.g.,
HMGCoA Lyase
Hydroxymethylglutaryl - CoA AcetylCoA Acetoacetate
(Class 5)=Isomerases move groups within a molecule, without changing the gross composition of the
substrate.e.g.,
Phosphotrioseisomerase
Dihydroxyacetonephosphate Glyceraldehyde 3 Phosphate
(Class 6)=Ligases (Synthetases): The ligation reactions catalyzed by are energy-dependent and are
therefore always coupled to the hydrolysis of nucleoside triphosphates.e.g.,
FattyAcylCoASynthetase
ATP FattyAcid AMP FattyAcyl CoA
Allosteric Enzymes:
An allosteric enzyme is one in which the binding of a ligand to one site affects the binding
properties of another site on the same protein.
Q/What are the properties of allosteric enzymes:
A/
1. Allosteric enzymes are usually present in oligomeric form (2–12 subunits). Some are catalytic subunits and
some are regulatory subunits.
Catalytic subunits bind with substrate
Regulatory subunits bind the allosteric effectors CTP and ATP.
2. In most allosteric enzymes, the affinity of allosteric enzymes is not constant, but depends on the type and
concentration of the substrate.
3. Allosteric enzymes have multiple substrate-binding sites of a cooperative effect. (If the interacted molecule is
the same substrate =Homotropic interaction (always cooperative). If another molecule= Heterotropic
interaction(may be cooperative or antagonistic)
4. Allosteric enzymes can occur in various conformations, which have different catalytic properties and whose
proportion of the total number of enzyme molecules is influenced by substrates and other ligands (inhibitors
or activators).
5. Plot of Vo against [S] is distinctly S-shaped (sigmoidal), which cannot be described using the Michaelis
model.
6. Allosteric enzymes can be present in two conformations: the less active T state (for ―tense‖) and the more
active R state (for ―relaxed‖). Substrates and effectors influence the equilibrium between the two states, and
thereby give rise to sigmoidal saturation behavior.
Figure: Catalytic sites (C ) will be ready to bind to substrate after Figure: Changes in conformation near heme on O2 binding
binding of regulatory parts (R) with a positive modulator to deoxyhemoglobin
Cofactors: serve as recyclable shuttles—or group transfer reagents—that transport many substrates from
their point of generation to their point of utilization. It binds in a transient (cofactors associate reversibly
with enzymes or substrates), 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.
Chemical moieties transported by coenzymes include methyl groups (folates), acyl groups (coenzyme A),
and oligosaccharides.
Note: Many Coenzymes, Cofactors, & Prosthetic Groups Are Derivatives of B Vitamins such as
Nicotinamide and riboflavin.
Q/what is the difference between coenzymes and prosthetic group?
A/ the coenzymes need to move from enzyme to enzyme to carry out their function. While prosthetic group
bind to enzymes. To be active for example: ATP is converted to ADP during hexokinase action but another
phosphate transfer (catalyzed by another enzymes.) is required to convert ADP back to ATP.
Apoenzyme + Prosthetic group or Cofactor HoloEnzyme
(Inactive) (Active)
THE ACTIVE SITE:
The active site is a cleft or pocket of three dimensional small portion of enzyme molecule at which the
substrate contact with. The properties of the active sites:
1. Take small size of the enzyme molecules.
2. It’s a group of sequences of a.a. folded by three dimensional structure.
3. It represents the catalytic site of the enzyme.
4. substrate binds with the enzyme by multiple weak attractions at the active site.
5. Active sites are cleft or cervices at which the substrate binds with.
1/ Effect of temperature:
There is an optimal temperature at which the reaction is most rapid. Above or below this
temperature, the reaction rate decreases sharply, generally due to heat denaturation of the enzymes.
The optimal temperature approximates those of the environment of the cell. the enzymes of human
has the optimal temperature of body =37.5°C
When temperature increases, there is consequently loss of secondary, tertiary structure & parallel
loss of biological activity.
Note: IF a cell extract having catalytic activity loses this activity when boiled (denaturized), the catalyst
probably was an enzymes.
2/PH: Every enzyme has an optimum pH (or pH range) at which it has maximal activity.
pH changes affects the ionic state of the enzymes & substrate. Optimal activity of enzymes is generally
observed between pH values of (5-9). However, a few enzymes e.g. pepsin are active at pH values
outside this range. The shape of pH activity shape (Figure) determined by these factors
1/ Enzymes denaturation at high or low pH values
2/ Effects on the charged state of the substrate or enzymes.
The charge changes may affect by changing the structure or by changing the functional groups in
substrate binding or catalysis.
Figure: Effect of temperature on the enzymes activity Figure: The pH optima of some enzymes.
5/ Oxidation: sulfhydryl (-SH) groups of many enzymes are essential for enzymes activity. Oxidation of
these (SH) groups forming disulfide linkages (S-S) leads to conformational changes.
e.g: Dehydrogenates enzymes are active when (-SH) present in reduced form and inactive in (S-S)
from
(oxidized form).
e.g. Ribonuclease are active when (-S-S-) present & inactive when (-SH) present in the
enzymes.
6/Radiation: Enzymes are highly sensitive to short wavelength (high energy) such as (UV, X, β, or γ –
rays). This due to oxidation of the enzymes by peroxides formed by high energy radiation.
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
Inhibition of enzyme Catalyzed reaction:
Inhibitors are chemicals that act by combining with S, cofactor or various forms of the
one enzyme. There are two types of inhibitors:
1/ Irreversible inhibitors: inhibitor binds with substrate to produce undissociable
complex and so inactivate enzymes.
E 1E1
e.g.: iodoacetamide bind with essential (- SH) group and in actirate enzymes
Enz SH ICH 2 CONH 2 Enz S CH 2 CONH 2 HI
E.g. organophosphorous compounds inhibits cholinesterase by binding with (-OH) group
of serine side chain.
a-Competitive inhibition: inhibitors bind with the active site. A competitive inhibitor
competes with the substrate for the active site of an enzyme.
In the presence of a competitive inhibitor, the Michaelis-Menten equation becomes:
where and
In Linweaver-Burk plot: alter its slop but do not change intercept
e.g: malonate is a competitive inhibitor of succinate dehydrogenase
b-Mixed inhibitor: also binds at a site distinct from the substrate active site, but it binds to either E
or ES. The rate equation describing mixed inhibition is
c-Uncompetitive inhibition: inhibitors bind with the complex (ES). In the presence of an
uncompetitive inhibitor, the Michaelis-Menten equation is altered to
Where
In lineweaver-Burk plot: alter intercept and not affect the slope.
Note: In practice, uncompetitive and mixed inhibition are observed only for enzymes with two or more
substrates— say, S1 and S2—and are very important in the experimental analysis of such enzymes.
The experimentally determined variable α Km, the Km observed in the presence of the inhibitor, is
often called the ―apparent‖ Km.