Mechanisms of Enzymes

Question 1

Proteases
•Proteases are enzymes that cleave ___ ____ by ____
•The resonance stabilized peptide bond is ___ ___ ___ (high activation energies) requiring protease enzymes to cleave the bond.
•Several proteases are involved in the break down of proteins in the ___ ___ of higher organisms.

Answer 1

•Proteases are enzymes that cleave peptide bonds by hydrolysis.
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•The resonance stabilized peptide bond is kinetically very stable (high activation energies) requiring protease enzymes to cleave the bond.
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•Several proteases are involved in the break down of proteins in the digestive system of higher organisms.

Question 2

Chymotrypsin
•Cleaves peptide bonds selectively on the ____ terminal side of the ___ ____ amino acids such as ____ ____ ____ ____. Determines specificity of the enzyme.
•Chymotrypsin uses _____interactions as a catalytic strategy to stabilize the ___ _____

• Consists of _ peptide chains linked by ___ ____
• Initially synthesized as a ___ ____ ______, activated by cleavage to yield __ chains.
• Overall forms a _____structure.
• Posses a catalytic ___residue (______) in the active site located on the surface of protein.
• Serine residue is ________ to _____ which in turn is_____ to ______ in the active site.
• The 3 amino acids in the active site are known as the ________ ____.

Answer 2

• Cleaves peptide bonds selectively on the carboxyl terminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine, and methionine. Determines specificity of the enzyme.
• Chymotrypsin uses covalent interactions as a catalytic strategy to stabilize the transition state.
• Consists of 3 peptide chains linked by disulfide bonds.
• Initially synthesized as a single chain chymotrypsinogen, activated by cleavage to yield 3 chains.
• Overall forms a spherical structure.
• Posses a catalytic serine residue (Ser 195) in the active site located on the surface of protein.
• Serine residue is H-bonded to Histidine, which in turn is H-bonded to Aspartic acid in the active site.
• The 3 amino acids in the active site are known as the catalytic triad.

Question 3

Catalytic Mechanism of Chymotrypsin

• Formation of a____ _____ ____ intermediate carrying a ____charge.
• Negative charge is stabilized by ___ ____
• This site is known as the____ ____
• Stabilizes the transition state. (lowers the activation energy of the reaction)

Answer 3

• Formation of a unstable tetrahedral covalent intermediate carrying a negative charge.
• Negative charge is stabilized by electrostatic interactions.
• This site is known as the oxyanion hole.
• Stabilizes the transition state. (lowers the activation energy of the reaction)

Question 4

Specificity of Chymotrypsin

•____ pocket (known as the _ pocket) enables residues ___ ___ ___ and ___ to __ in and ____ the peptide bond into the ___ ____ for ____
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•This determines the ____ of the enzyme.

Answer 4

•Hydrophobic pocket (known as the S pocket) enables residues tryptophan, tyrosine, phenylalanine, and methionine fit in and orient the peptide bond into the active site for cleavage.
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•This determines the specificity of the enzyme.

Question 5

Specificity of other serine proteases

• Trypsin cleaves at the peptide bond after residues with ___, ____ charged side chains (____ ____)
• Elastase cleaves at the peptide bond after amino acids with ____ side chains (___ ____).
• S pockets of these enzymes defines the specificity.
• In trypsin, ___# is present at the bottom of the S pocket in place of a serine residue in chymotrypsin. The aspartate residue ____ and___ a positively charged arginine or lysine residue in the substrate.
• In elastase, two residues at the top of the pocket in chymotrypsin and trypsin are replaced with ____(#).
• These residues close off the mouth of the pocket so that only ____ ____ chains may enter.

Answer 5

• Trypsin cleaves at the peptide bond after residues with long, positively charged side chains (arginine and lysine).
• Elastase cleaves at the peptide bond after amino acids with small side chains (alanine and serine).
• S pockets of these enzymes defines the specificity.
• In trypsin, Asp 189 is present at the bottom of the S pocket in place of a serine residue in chymotrypsin. The aspartate residue attracts and stabilizes a positively charged arginine or lysine residue in the substrate.
• In elastase, two residues at the top of the pocket in chymotrypsin and trypsin are replaced with valine (Val 190 and Val 216).
• These residues close off the mouth of the pocket so that only small side chains may enter.

Question 6

Catalytic Mechanisms of Cysteine, Aspartyl and Metalloproteases

• Cysteine proteases- ____, activated by a ____ , plays the role of the nucleophile that attacks the peptide bond. Example - ____
• Aspartyl proteases – A ____ of ______ residues that act together to allow a ____ ____ to attack the peptide bond. Example -____
• Metalloproteases - ___ ___ activates a ____ ____to act as a nucleophile to attack the peptide ___ ___. Example - ______

Answer 6

•Cysteine proteases- Cysteine, activated by a histidine , plays the role of the nucleophile that attacks the peptide bond. Example - Caspases•Aspartyl proteases – A pair of aspartic acid residues that act together to allow a water molecule to attack the peptide bond. Example - Renin
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•Metalloproteases - metal ion activates a water molecule to act as a nucleophile to attack the peptide carbonyl group. Example - Angiotensin converting enzyme (ACE)
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Question 7

Use of Protease Inhibitors in Disease

• Protease inhibitors are similar to the structures of the ___ ____
• These inhibitors used as drugs must be specific for ___ enzyme without _____
• Example - ____, inhibitor of the _____ ____

Answer 7

• Protease inhibitors are similar to the structures of the substrate peptides.
• These inhibitors used as drugs must be specific for one enzyme without inhibiting other proteins within the body.
• Example - Crixivan, inhibitor of the HIV protease.

Question 8

Summary

• The ___ ____is important for the function of serine proteases.
• The ___ ____ define the specificity of each serine protease.
• Catalytic mechanisms of cysteine, aspartyl and metalloproteases ___ from one another.

Answer 8

• The catalytic triad is important for the function of serine proteases.
• The S pockets define the specificity of each serine protease.
• Catalytic mechanisms of cysteine, aspartyl and metalloproteases differ from one another.