catalytic strategies. basic catalytic principles what is meant by the binding energy as it relates...
TRANSCRIPT
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?
O2 N OCCH3
O
O2 N O-
CH3 CO-O
p-Nitrophenylacetate
p-Nitrophenolate
Step 1 E +
E-OCH3
O
+
Step 2 E-OCH3
O+ H2 O E +
Enzyme
An acyl-enzymeintermediate
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
Chymotrypsinmechanism of action
Chymotrypsinmechanism of action
Chymotrypsinmechanism of action
Chymotrypsinmechanism of action
Chymotrypsinmechanism of action
Chymotrypsinmechanism of action
Chymotrypsinmechanism of action
Chymotrypsinmechanism of action
Chymotrypsinmechanism 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
• What ensures specificity of the EcoRV reaction?– Inverted repeats of recognition site creates a twofold
rotational symmetry
Restriction Enzymes
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 P-loop NTPase domains.