Download Mechanism of Enzyme Action

Dr. Ketki K,
MBBS, MD
Assistant Professor
Department of Biochemistry
Mechanism of Enzyme Action
• Prerequisites for catalysis by enzymes:
Lowering of activation energy/
Transition state theory:
? Activation energy: Energy required by the
reactant to undergo the reaction is known as
activation energy (OR)
energy required to convert all molecules of
reacting substance from ground state to
transition state
The reactants when heated,
attain the activation energy
Lowering of activation energy/
Transition state theory:
• Enzyme(Catalyst) lowers the activation
energy
so that, reaction proceeds at a lower
temperature
• In other words, enzymes lower the energy
barrier of reactants, so that reaction goes
faster at body temperature(below40 degree C)
A+B
AB*
C+D
E+S
ES
E+P
Reactants
Transition
state
Product
Enzyme Stabilizes Transition State
Energy change
EST
S
ES
EP
Reaction direction
T = Transition state
P
Energy decreases (under catalysis)
Energy required (no catalysis)
ST
What’s the difference?
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166
Mechanism of enzyme action
Active Site
• Small region on enzyme, at which
substrate binds and participates in
catalysis
Salient features:
• 3D structure
• Existence is due to tertiary structure of protein resulting
in three dimensional native conformation
• Cleft / crevices / pockets occupy a small portion of big
enzyme
• Flexible
• Substrate binding site and catalytic site
• Substrate binds at the active site non-covalently,
• Forces are hydrophobic in nature
• The amino acids participate in making & breaking the bonds
at the active site are c/a catalytic residues
• Amino acids far apart from each other in linear amino acid
sequence
Amino acids at catalytic site
Name of the enzyme
Chymotrypsin
Trypsin
Important amino acids
at the catalytic site
His (57),Asp(102),Ser(95)
Alkaline phosphatase
Serine, Histidine,
Arginine, Lysine
Serine
Carbonic anhydrase
Cysteine
Hexokinase
Histidine
Different Enzyme Models/
theory
• Michaelis and Menten theory
• Fischer’s Lock and key model
• Koshland’s Induced Fit Model
Michaelis and Menten theory/
enzyme substrate complex
theory
• Example of alkaline Phosphatase:
active center of it contains serine residues
E- Serine-OH + Glucose-6-P
E-Serine-O-P + Glucose
E-Serine-O-P
E-Serine-OH + Pi
Glucose-6-P
Glucose + Pi
Fischer’s Lock and Key Model
P
S
+
+
S
P
E
+
S
ES complex
E
+
P
Koshland’s Induced Fit Model
P
S
S
P
E
+
S
ES complex
E
+
P
Active site
1) Defination
2) Salient features
3) Aminoacids at active site/catalytic sitetable
4) Different enzyme models/theory
Mechanism of Catalysis
•
Acid - Base catalysis : In this process
the enzyme gives or takes H+ to bring
about catalysis.
(At physiological pH, histidine is the
most important amino acid, the
protonated form of which functions as
an acid and its corresponding
conjugate as a base)
 Example of acid base catalysis:
Action of ribonuclease
Histidine residues 12 and 119 at the active
site of ribonuclease function as acid &
base respectively and donate proton &
accept proton respectively.
• Substrate strain
• Substrate binding to active site induces
a strain to the substrate during which
the energy level of substrate is raised,
leading to a transition state. Eg:
Lysozyme
• Covalent catalysis
• The negatively charged or positively
charged groups are present at the
active site of the enzyme attack the
substrate that results in the covalent
binding of the substrate to the enzyme
Eg: Serine proteases
• Proximity catalysis(proximity effect/entropy
effect)
• Reactant should come in close proximity
to enzyme, for appropriate catalysis to
occur
• Higher the concentration of substrate
molecules, greater will be the rate of
reaction.
Product substrate orientation theory
Enzyme has appropriate three
dimensional structure to keep the
substrates in a specific orientation, such
that reactive groups come to physical
apposition.
Eg: Glucose + ATP = Glucose-6-P
Enzyme Specificity
Group Specificity
One enzyme can catalyze the same reaction
on a group of structurally similar compounds
Eg: Hexokinase can catalyze phosphorylation of
glucose, galactose & mannose
Absolute specificity
• Some enzymes are absolutely specific
Eg:1)Glucokinase: Phosphorylates only glucose
Glucose + ATP ---------------→ Glucose – 6 –
phosphate + ADP
2)Galactokinase: Phosphorylates only
galactose
Galactose + ATP ---------------→ Galactose – 1
– phosphate + ADP
3) Urea is the only substrate for urease
Optical specificity
Enzymes act only on one isomer
L – amino oxidase
L-amino acid ------------------→ α – keto acid + NH3
FMN
FMNH2
D – amino acid oxidase
D–amino acid ------------------→ α - keto acid + NH3
FAD
FADH2
Bond specificity
• Proteolytic enzymes show bond specificity
Eg: Trypsin can hydrolyze peptide bonds
formed by carboxyl group of arginine or
lysine residues in any protein
Attention Check !!
A. The active site is
(1) the enzyme
(2) a section of the enzyme
(3) the substrate
B. In the induced fit model, the shape of the
enzyme when substrate binds
(1) stays the same
(2) adapts to the shape of the substrate
Answers
A. The active site is
(2) a section of the enzyme
B. In the induced fit model, the shape of
the enzyme when substrate binds
(2) adapts to the shape of the
substrate
Attention Check !!
A. Enzyme trypsin is specific for
(1) Arginine and Lysine
(2) Phenylalanine and Tryptophan
(3) Alanine and Glycine
B. The reaction that occurs in substrate
strain is
(1) Conversion to transition state
(2) Chemical reaction
Answers
A. Enzyme trypsin is specific for
(1) Arginine and Lysine
B. The reaction that occurs in substrate strain is
(1) Conversion to transition state
CLINICAL NOTE
• Because most vitamins function as coenzymes,
the symptoms of vitamin deficiencies reflect the
loss of specific enzyme activities that depend on
the coenzyme form of the vitamin.
• Thus, drugs and toxins that inhibit proteins
required for coenzyme synthesis (e.g., vitamin
transport proteins or biosynthetic enzymes) can
cause the symptoms of a vitamin deficiency.
• This type of deficiency is called a functional
deficiency, whereas an inadequate intake is called
a dietary deficiency.
• Most coenzymes are tightly bound to their
enzymes and do not dissociate during the course
of the reaction.
• However, a functional or dietary vitamin deficiency
that decreases the level of a coenzyme will result
in the presence of the apoenzyme in cells (an
enzyme devoid of coenzyme).
• Ethanol is an “antivitamin” that decreases the
cellular content of almost every coenzyme. For
example, ethanol inhibits the absorption of
thiamine, and acetaldehyde produced from ethanol
oxidation displaces pyridoxal phosphate from its
protein-binding sites, thereby accelerating its
degradation.
CLINICAL NOTE
• Many alcoholics develop thiamine deficiency
because alcohol inhibits the transport of thiamine
through the intestinal mucosal cells.
• In the body, thiamine is converted to thiamine
pyrophosphate (TPP). TPP acts as a coenzyme in
the transamination & decarboxylation reactions of
amino acid metabolism and in the utilization of
pentose phosphates in the pentose phosphate
pathway.
• As a result of thiamine deficiency, Dysfunction
occurs in the central and peripheral nervous
system and other organs.
CLINICAL NOTE
• In humans, most of ingested ethanol is oxidized to
acetaldehyde in the liver by alcohol
dehydrogenase (ADH)
• ADH is active as a dimer, with an active site
containing zinc present in each subunit. The
human has at least seven genes that encode
isozymes of ADH, each with a slightly different
range of specificities for the alcohols it oxidizes.
• The acetaldehyde produced from ethanol is highly
reactive, toxic, and immunogenic. In patients with
chronic alcoholism, acetaldehyde is responsible
for much of the liver injury associated with chronic
alcoholism.
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