Catalytic Strategies

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Catalytic Strategies
Basic Catalytic Principles
• What is meant by the binding energy as it relates
to enzyme substrate interactions?
– free energy released by formation of weak interactions
between enzyme and substrate
• How is binding energy used in catalysis?
– establishes substrate specificity
– increases catalytic efficiency
– promotes structural changes in enzyme and substrate
Basic Catalytic Principles
• What are the four strategies used by
enzymes to carry out catalysis?
– covalent catalysis
• functional groups in enzyme act as nucleophile
– OH groups of serine
– SH groups of cysteine
– Imidazole group of histidine
• example - chymotrypsin
Basic Catalytic Principles
– general acid-base catalysis
• enzymes provide functional groups that act as
proton donors or acceptors
– amino groups
– carboxyl groups
– sulfhydryl groups
• Example – lysozyme (donates proton)
Basic Catalytic Principles
– metal ion catalysis
• metal ion acts as electrophilic catalyst
– stabilizes negative charge on intermediate
• metal ion may generate a nucleophile by increasing
acidity of nearby molecule
– example – carbonic anhydrase
• metal ion may bind to substrate and increase binding
energy
– example – NMP kinases
Basic Catalytic Principles
– catalysis by approximation
• binding to enzyme brings two substrates together
– example – NMP kinases
• binding to enzyme orients substrate
– susceptible bond is close to catalytic groups of active site
– orbital steering (Koshland and Storm)
Proteases
• What are proteases?
– protein degrading enzymes
• Why are proteases important in biological
systems?
– recycling of amino acids
– digestion of proteins in diet
– Enzyme regulation
Proteases
• Describe the reaction catalyzed by
proteases.
• Why is it so difficult to break a peptide
bond?
Chymotrypsin
• What is the nature of the reaction is catalyzed by
chymotrypsin?
Chymotrypsin
• What catalytic strategy does chymotrypsin
use?
– covalent catalysis
• nucleophilic group in enzyme attacks unreactive
carbonyl group of substrate forming covalent bond
• Which group of enzyme acts as the
nucleophile?
– serine 195
Chymotrypsin
• How was it possible to identify the specific
functional group involved in catalysis?
Chymotrypsin
• What are the two steps involved in the action of
chymotrypsin?
O
Step 1
E
+
Enzyme
O2 N
OCCH 3
p-Nitrophenylacetate
O
E-OCH3
An acyl-enzyme
intermediate
O
Step 2
E-OCH3 +
H2 O
+ O2 N
O-
p-Nitrophenolate
O
E + CH3 CO -
Chymostrypsin
• Can see two stages of chymotrypsin catalysis
Chymotrypsin
• Three-dimensional
structure of enzyme
has helped to reveal its
catalytic mechanism
Chymotrypsin
• Three amino acid residues are involved in
catalysis: serine 195, histidine 57, aspartate 102
Chymotrypsin
mechanism of action
Chymotrypsin
mechanism of action
Chymotrypsin
mechanism of action
Chymotrypsin
mechanism of action
Chymotrypsin
mechanism of action
Chymotrypsin
mechanism of action
Chymotrypsin
mechanism of action
Chymotrypsin
mechanism of action
Chymotrypsin
mechanism of action
• Unstable tetrahydral
intermediate is
stabilized by
interactions with NH
groups from protein at
oxyanion hole
Chymotrypsin
• What accounts for the
preference of this
enzyme cleaving
peptide bonds adjacent
to residues with large
hydrophobic side
chains?
Serine Proteases
• Other proteases use same catalytic triad
– trypsin
• cleaves at peptide bond after residue with long,
positively charged side chain
– elastase
• cleaves at peptide bond after residue with small side
chains
• Specificity depends upon residues in pocket
Serine Proteases
• Subtilisin from Bacillus amyloliquefaciens also
uses catalytic triad and oxyanion hole
Serine Proteases
• Carboxypeptidase II which has a very different
structure also uses catalytic triad and oxyanion
hole
Serine Proteases
• Presence of similar active sites in different
protein families is a result of convergent
evolution.
• What does this tell us about this mechanism
for the hydrolysis of peptides?
• What is site-directed mutagenesis and how
is it used?
Other Proteases
• What are cysteine, aspartyl and
metalloproteases?
– Cysteine proteases – cysteine residue acts as
nucleophile
• Papain
– Aspartyl proteases – pair of aspartate residues
act to enable water molecule to attack peptide
bond
• renin
Other Proteases
– Metalloproteases – active site contains a metal
ion that activates water to act as a nucleophile
• metal is usually zinc
• Carboxypeptidase A
Protease Inhibitors
• Several useful drugs
– Catopril – ACE inhibitor
• regulator of blood pressure
– Crixivan – HIV protease
• AIDS treatment
Carbonic Anhydrases
• What is the reaction catalyzed by these
enzymes?
– CO2 + H2O  H2CO3  HCO3- + H+
• Where does this reaction take place?
Carbonic Anhydrases
• What metal ion is associated with these enzymes
and where is it bound to the enzyme?
Carbonic Anhydrases
• How does the zinc complex facilitate the hydration of
carbon dioxide?
– Binding of water to zinc reduces pKa of water from 15.7 to 7 and
creates a hydroxide ion that can act as a nucleophile
Carbonic Anhydrases
• Mechanism of action
Carbonic Anhydrases
• What enables these enzymes to be extremely
effective catalysts?
– Proton shuttle mechanism
Restriction Enzymes
• What are restriction endonucleases and how
have they been used by bacterial cells?
– Enzymes that cleave DNA
– Protection against invading viruses
• What is cognate DNA?
– DNA containing recognition sites
Restriction Enzymes
• How do bacteria protect their own DNA from
cleavage?
Restriction Enzymes
• What bond is cleaved by these enzymes?
– Bond between 3! oxygen atom an phosphorous atom
Restriction Enzymes
• What is the mechanism of this reaction?
– direct attack of water as a nucleophile on
phosphorous
• Why is Mg+2 needed for activity?
– Mg+2 helps position water molecule to attack
phosphate
– Mg+2 along with aspartate residue helps to
deprotinate water molecule
Restriction Enzymes
Restriction Enzymes
• What ensures specificity of the EcoRV reaction?
– Inverted repeats of recognition site creates a twofold
rotational symmetry
Restriction Enzymes
• G and A bases at 5! End form hydrogen bonds with
residues from two loops of enzyme causing
distortion of DNA and bonding with Mg+2
Restriction Enzymes
• How does methylation protect host DNA from
restriction enzymes?
Restriction Enzymes
• What is horizontal
gene transfer and how
do this relate to the
presence of restriction
enzymes in bacteria?
Restriction Enzymes
• Different bacteria share common gene sequences
and a similar active site conformation
NMP Kinases
• What reaction do nucleoside
monophosphate kinases catalyze?
– transfer of phosphate group from a nucleoside
triphosphate to a nucleoside monophosphate
• Example of NMP kinase?
– adenylate kinase
NMP Kinases
NMP Kinases
• What structural features does this family of
enzymes have in common?
NMP Kinases
• What role does Mg+2 or Mn+2 play in the catalytic
mechanism of these enzymes?
NMP Kinases
• How do we know that
binding between
enzyme and substrate
involves an induced fit
mechanism?
• What catalytic strategy
is used by this
enzyme?
NMP Kinases
• Many important
proteins contain Ploop NTPase
domains.
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