Enzymes Flashcards
1 molecule of an enzyme can act upon
About 1000 molecules of substrate per min
Lack of enzymes will lead to
Block in metabolic pathways causing inborn errors of metabolism
Characteristics of enzymes (5)
Almost all enzymes are proteins. Enzymes follow the physical and chemical reactions of proteins.
They are heat labile
Water soluble
Pptd by protein ppting reagents (ammonium sulfate or trichloroacetic acid)
Contain 16% weight as nitrogen
IUBMB classification of enzymes in order
Oxidireductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
Translocases
Enzymes are grouped into
7 major classes
Enyme classification.
NAME
EXPLANATION
ENZYME EXAMPLE
REACTION
2 EXAMPLES
OXIDIREDUCTASES
This group of enzymes will catalyse oxidation of
one substrate with simultaneous reduction of
another substrate or co-enzyme.
This may be
represented as
AH2 + B ————-→ A + BH2
for example,
Alcohol + NAD+ —-→Aldehyde+NADH + H+
The enzyme is Alcohol dehydrogenase;
IUB
name is Alcohol-NAD-oxidoreductase; Code
number is EC.1.1.1.1.
Oxidoreductases may also
oxidise substrates by adding oxygen, e.g.
oxidases, oxygenases and dehydrogenases
TRANSFERASES
This class of enzymes transfers one group
(other than hydrogen) from the substrate to
another substrate.
This may be represented as
A-R + B → A + B-R
,
For example,
Hexose + ATP → Hexose-6-phosphate + ADP
The name of enzyme is Hexokinase and
systematic name is ATP-Hexose–6-phosphate-
transferase.
HYDROLASES
This class of enzymes can hydrolyse ester, ether,
peptide or glycosidic bonds by adding water and
then breaking the bond.
Acetyl choline + H2O ——–→ Choline + acetate
The enzyme is Acetyl choline esterase or
Acetyl choline hydrolase (systematic).
All
digestive enzymes are hydrolases.
LYASES
These enzymes can remove groups from
substrates or break bonds by mechanisms other
than hydrolysis.
For example,
Fructose-1,6-bisphosphate ——-→
Glyceraldehyde-3-phosphate
+dihydroxy acetone phosphate
The enzyme is aldolase
Subclass : hydratase, hydration of a double bond or adding water to a double bond
ISOMERASES
These enzymes produce optical, geometric
or positional isomers of substrates.
Racemases,
epimerases, cis-trans isomerases are examples.
Glyceraldehyde-3-phosphate —–→
Di-hydroxy-acetone-phosphate
Enzyme is Triose phosphate isomerase.
LIGASES
These enzymes link two substrates together,
usually with the simultaneous hydrolysis of ATP
For example,
Acetyl CoA + CO2 + ATP → Malonyl CoA + ADP +
Pi
Enzyme is Acetyl CoA carboxylase.
TRANSLOCASES (3)
Catalyse the translocation of ions and other molecules across membranes (from one side of the membrane to other side)
Some of em use E of hydrolysis of ATP for the translocation and were previously included with Atpases(class3)
There are specific transl for transferring H ions inorganic anions cations aas carbohydrates and other compounds
Enzyme inhibition and clinical significance (6). TYPES
Competitive
Non competitive
Uncompetitive
Suicide inhibition
Allosteric regulation/ inhibition
Feedback inhibition
COMPETITIVE INHIBITION
- Explain
- Reaction velocity is decreased
- Structural analog
- Reversible
- From the graphs
1.Here inhibitor molecules are competing with
the normal substrate molecules for binding to
the active site of the enzyme,because the
inhibitor is a structural analog of the substrate.
E + S E-S ——-→ E + P
E + I E-I
- Since E-I (enzyme–inhibitor complex) can react
only to reform the enzyme and inhibitor, the
number of enzyme molecules available for E-S
formation is reduced. Suppose 100 molecules
of substrate and 100 molecules of inhibitor are
competing for 100 molecules of the enzyme.
So, half of enzyme molecules are trapped by
the inhibitor and only half the molecules are
available for catalysis to form the product.
Since effective concentration of enzyme is
reduced, the reaction velocity is decreased. - competitive inhibition, the inhibitor will be a
structural analog of the substrate. There will
be similarity in three dimensional structure
between substrate (S) and inhibitor (I). For
example, the succinate dehydrogenase
reaction is inhibited by malonate - Competitive inhibition is usually reversible.
Or, excess substrate abolishes the inhibition.
In the previous example of 100 moles of E and
100 moles of I, if 900 moles of S are added,
only 1/10th of enzyme molecules are attached
to inhibitor and 90% are working with substrate.
Thus 50% inhibition in the first example is now
decreased to 10% inhibition - From the graphs, it is obvious that in the case
of competitive inhibition, the Km is increased
in presence of competitive inhibitor. Thus
competitive inhibitor apparently increases the
Km. In other words, the affinity of the enzyme
towards substrate is apparently decreased in
presence of the inhibitor.
But Vmaxis not changed.
NONCOMPETITIVE INHIBITION
Definition (5)
Examples
Irreversible
Graph(2)
A variety of poisons, such as iodoacetate,
heavy metal ions (lead, mercury) and oxidising
agents act as irreversible non-competitive inhibitors.
There is no competition between S and I.
Also known as mixed inhibition
The inhibitor usually binds to a different domain on the enzyme other than the substrate binding site.
Since these inhibitors have no structural resemblance to the substrate an increase in S conc generally does not relieve this inhibition.
Examples :
CN inhibits cytochrome oxidase
Fluoride will remove Mg and Mn ions and so will inhibit the enzyme enolase and consequently glycolysis
Iodoacetate inhibits E having SH groups in their active centres
BAL - British Anti Lewisite ; dimercaprol is used as an antidote for heavy metal poisoning. The heavy metals acts as enzyme poisons by reacting with the SH group. Bal has several SH groups with which the heavy metals ions can react and thereby their posionous effects are reduced.
The inhibitor combines with the enzymes by
forming a covalent bond and then the reaction
becomes irreversible. The velocity (Vmax) is
reduced. But Km value is not changed,
because the remaining enzyme molecules
have the same affinity for the substrate.
UNCOMPETITIVE INHIBITION
Definition (3)
Examples (2)
Inhibitor does not have any affinity for free enzyme
Inhibitor binds to E-S complex but not to free E
Both Vmax and Km are reduced
Examples:
Inhibition of placental alkaline phosphatase(Regan isoenzyme) by phenylalanine
Anticonvulsant valproate and anticancer drug camptothecin are un inhibitors
Valp inhibits it’s own mechanism
Campto blocks cell division by inhibiting topoisomerase1
SUICIDE INHIBITION
Definition (3)
Process(3)
It is a special type of irreversible inhibition of enzyme activity
Also known as mechanism based inactivation
I makes use of Es own reaction mechanism to inactivate it.
Structural analog is converted to a more effective inhibitor with the help of E to be inhibited
S like compound initially bind with the E and the first few steps of pathway are catalyzed
This new product irreversiblly binds to the E and inhibits further reactions
Examples of suicide inhibition (2)
Allopurinol which is oxidised by xanthine oxidase to alloxanthine that is a strong I of xanthine oxidase
Anti inflammatory action of aspirin
Arachidonic acid is converted to prostaglandin by E cyclooxygenase. Aspirin acetylates a serine residue in the active center of cyclo thus prostaglandin synthesis is inhibited and so inflammation subsides
ALLOSTERIC REGULATION
Inhibition (6)
Regulation (6)
- The inhibitor is not a substrate analog.
- It is partially reversible, when excess substrate
is added. - Km is usually increased.
- Vmax is reduced.
- The effect of allosteric modifier is maximum at
or near substrate concentration equivalent to
Km.When an inhibitor binds to the
allosteric site, the configuration of catalytic site
is modified such that substrate cannot bind
properly. - Most allosteric enzymes possess quaternary
structure. They are made up of subunits, e.g.
Aspartate transcarbamoylase has 6 subunits
and pyruvate kinase has 4 subunits.
Allosteric enzymes has one catalytic site where the substrate binds and another allosteric site where the modifier binds. May or may not be physically adjacent
Regulatory molecule - positive modifier - binding of Rm enhances the activity of E (allosteric activation
Opp
Positive cooperativity - binding of S to one of the subunits increase binding to other subunits
Negative cooperativity
Key enzymes:
Body uses allosteric E for regulating metabolic pathways.
Such a regulatory E in a particular pathway is called the Key E or Rate limiting E.
Eg phosphofructokinase.
Fructose 6po4 + Atp gives fructose 16bis + ADP
Ala synthase
Suucinyl coa + glycine gives delta ala
Examples of ALLOSTERIC enzymes
Enzyme
allosteric inhibitor
allosteric activator
1. ALA synthase heme
- Aspartate trans-carbamoylase
CTP
ATP - HMGCoA-reductase
Cholesterol - Phospho-, fructokinase
ATP, citrate
AMP, f-2,6-p - Pyruvate carboxylase
ADP
AcetylCoA - Acetyl CoA- carboxylase
Acyl coA
Citrate - Citrate synthase
ATP - Carbamoyl phosphate synthetase I
NAG - Carbamoyl phosphate synthetase II
UTP
Positive modifier / allosteric activation
The binding of regulatory molecule enhance the activity of E
Positive cooperativity
Binding of substrate to one of the subunits of E may enhance substrate binding by other subunits.
Shape of curve allosteric
Sigmoid shape
FEEDBACK INHIBITION
Activity of the E is inhibited by the product of the reaction
End product inhibition of biosynthetic pathways are also examples for this type of inhibition.
A ——-→ B ——–→ C ———→ D
In this pathway, if D inhibits E1, it is called end product inhibition
Examples of Feedback inhibition a (3)
First step in heme synthesis
EndP heme will allosterically inhibit ALA synthase
This E is the key E of heme synthesis
Regulation of E activity (5)
Induction
Repression
Covalent modification
Stabilization
Compartmentalisation
INDUCTION (3)
Effected through derepression
Inducer will relieve the repression on the operator site and will remove the block on biosynthesis of the E molecules
Examples
Lac operon
transaminases are induced by glucocorticoids.
Glucokinase is induced by insulin.
ALA synthase
is induced by barbiturates.
REPRESSION
Features (4)
Examples
Reduces the enzyme velocity
Repressor acts at gene level
Effect is noticeable only after a lag period of hours or days
No. Of E molecules is reduced in the presence of repressor molecule.
Key enzyme of heme synthesis Alas is autoregulatef by heme by means of repression.The
structural gene is transcribed and later translated to produce
the enzyme molecules. The transcription process starts at the
operator site when it is free. When heme is not available, this
operator site is open, and therefore the enzyme is being
synthesized. When heme is produced in plenty, heme acts as
the co-repressor and in combination with an apo-repressor,
heme will shut off the operator site. Now further production of
ALA synthase is stopped.
COVALENT MODIFICATION
Features (2)
Zymogen activation
Reversible protein phosphorylation
ADP ribosylation
The activity of enzymes may be increased or
decreased by covalent modification.
It means, either
addition of a group to the enzyme protein by a
covalent bond; or removal of a group by cleaving a
covalent bond.
Zymogen activation by partial proteolysis is an
example of covalent activation.
Addition or removal
of a particular group brings about covalent modification of Enzyme protein
This is a reversible reaction
STABILIZATION (3)
Examples
Enzyme molecules undergo usual wear and tear and
finally get degraded.
Such degradation if prevented can
lead to increased overall enzyme activity.
This is called
stabilization of enzyme.
Degradation of Tryptophan
pyrrolase is retarded by tryptophan.
Phospho fructo kinase
is stabilized by growth hormone.
Enzymes having SH-
groups (Papain, Urease, Succinate dehydrogenase) are stabilised by glutathione (G-SH)
COMPARTMENTALISATION (3)
Examples
The activity of enzymes catalysing the different steps in a
metabolic pathway may be regulated by compartmentalization of Es
Mito and cyto
Examples heme synthesis
Urea cycle
Gluconeogenesis
The intermediates have to be shuttled across
the mitochondrial membrane for this purpose which provides
a point where controls can be exerted.
Coenzymes (7)
- The protein part of the enzyme gives the necessary
three dimensional infrastructure for chemical
reaction; but the group is transferred from or
accepted by the co-enzyme. - The co-enzyme is essential for the biological
activity of the enzyme. - Co-enzyme is a low molecular weight organic
substance. It is heat stable. - Generally, the co-enzymes combine loosely with
the enzyme molecules. The enzyme and co-
enzyme can be separated easily by dialysis. - Inside the body, when the reaction is completed,
the co-enzyme is released from the apo-enzyme,
and can bind to another enzyme molecule. , the reduced co-
enzyme, generated in the first reaction can take
part in the second reaction. The coupling of
these two reactions becomes essential in
anerobic glycolysis for
regeneration of NAD+. - One molecule of the co-enzyme is able to convert
a large number of substrate molecules with the
help of enzyme. - Most of the co-enzymes are derivatives of vitamin B complex group of substances
Division of coenzymes
Those taking part in reactions catalyzed
by oxidoreductases by donating or
accepting hydrogen atoms or electrons.
Those co-enzymes taking part in rea-
ctions transferring groups other than
hydrogen.
First group(4)
Examples
the change occurring in the
substrate is counter-balanced by the co-enzymes.
Therefore, such co-enzymes may be considered
as co-substrates or secondary substrates.
the substrate lactate is
oxidized, and simultaneously the co-enzyme (co-
substrate) is reduced.
If the reaction is reversed,
the opposite effect will take place.
Other such examples are NADP–NADPH; FAD-
FADH2 and FMN–FMNH2.
Second group
These co-enzymes take part in reactions trans-
ferring groups other than hydrogen.
A particular
group or radical is transferred from the substrate
to another substrate.
Most of them belong to
vitamin B complex group.
Km value and it’s significance (4)
Km value is the substrate concentration expressed in mole/l at half maximal velocity
It denotes 50% E molecule bound with substrate molecules at that particular substrate conc.
Independent of E conc
Characteristic feature of a particular E for a specific substrate ( constant for an E)
Denotes the affinity of E for S. Lesser numerical value of Km affinity of Es for S is more
Isoenzymes(4)
Physically distinct forms of same E
Identical functions
Produced from diff tissues
Homomultimer / heteromultimer
Iso LDH(6)
Separation
Tetramer 4 subunits
Subunit - either Heart or muscle polypeptide chains
Both of them have same mol wt 32kD minor aa variations
5 isoenzymes
Represented as (5)
These 5 forms seen in all persons
Separated by cellulose acetate electrophoresis at pH 8.6
Bands are identified by adding the reactants containing nitro blue tetrazolium producing a color rxn which may be qhantitated by a scanner
Reaction of LDH
Normal value
Iso seen in
Pyruvate - lactate
In serum - 100-200 U/L
Flipped pattern of LDH
Normally ldh2 conc in blood > ldh1.
This pattern reversed in MI
During MI
Total LDH activity increased
H4 iso In 5-10 times more
LDH start to increase by 6-12 hour after athe MI reach a peak by 25-48 hours level go back to og by 6-8 days
100 × more in RBC than in plasma
Therefore minor amt of hemolysis results in a false positive test
Since total LDH increased in many conditions not used as a cardiac marker
Biomarkers
Naturally occurring molecule or gene or characteristic by which a particular pathological process can be identified.
Substance used as an indicator of the presence of a material of biological orgin or a physiological condition or process
A diagnostic indicator of (predisposition to) a medical condition.
Substance that can be used as an indicator of a particular disease state
Clinical lab test which is useful in detecting dysfunction of an organ
Cardiac markers
Of MI
For risk prediction
Of M stress or congestive cardiac failure
Early detection of acute MI
Cardiac tropinins ( TnI TnT) ss
CK -MB ss
Myoglobin sns
Indicator of congestive cardiac failure
Brain natriuretic peptide
Enzyme profiles in liver diseases
Alt
Alp
Ast
Ntp
Ggt
