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Basic Biochemistry > BB12 – Chymotrypsin and proteases > Flashcards

BB12 – Chymotrypsin and proteases Flashcards

0
Q

Common catalytic strategies

A
  1. covalent catalysis
  2. general acid-base catalysis
  3. metal-ion catalysis
  4. catalysis by approximation
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1
Q

Chymotrypsin

A
  • ester
  • digestive enzyme secreted by the pancreas
  • serine protease
  • carboxyl-terminal side of an aromatic or large hydrophobic residue
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2
Q

2 catalytic strategies of chymotrypsin

A
  • covalent catalysis

* general acid-base catalysis

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3
Q

Proteases cleave proteins by

A

hydrolysis – addition of water to a peptide bond

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4
Q

… inactivates chymotrypsin

A

di-isopropyl-fluorophosphate

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5
Q

Residue in the active site of chymotrypsin

A

Serine 195

also Histidine 57 and Aspartate 102

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6
Q

Covalent catalysis in chymotrypsin

A

nucleophile attacks carbonyl carbon of substrate

nucleophile covalently attached to substrate briefly

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7
Q

Kinetics of chymotrypsin monitored

A

• acts on substrate analog that forms a colored product
• p-Bitrophenolate turns yellow, can be measured
(chromogenic substrate)

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8
Q

Chymotrypsin in 2 phases

A
  1. acylation to form acyl enzyme intermediate – quick
  2. deacylation to regenerate free enzyme – slow
    • explained by formation of a covalently bound enzyme-substrate intermediate
    • use binding energy of substrate to accelerate the reaction
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9
Q

Serine held in active site by

A

disulfide bonds

• in a cleft next to histidine and aspartate = more reactive

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10
Q

Chymotrypsin shape

A

spherical

3 polypeptide chains linked with disulfide bonds

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11
Q

Catalytic triad

A
  • side chain of serine-195 hydrogen bonded to imidazole ring of histidine-57
  • NH of this ring hydrogen bonded to carboxylate group of aspartate-102
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12
Q

Alkoxide ion (O-)

A
  • H polarizes serine’s hydroxyl group
  • substrate -> histidine takes proton from serine
  • aspartate makes histidine a better proton acceptor
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13
Q

Stage 1 – acylation of the enzyme

A
  1. substrate binding
  2. nucleophilic attack of serine on the peptide carbonyl group
    • O on side chain of ser attacks carbonyl c
    • 4 atoms on C = tetrahedral intermediate
    • - charge on O = oxyanion hole – stabilized by interactions with NH groups from the protein
  3. collapse of the tetrahedral intermediate to form the acyl-enzyme
    • transfer of the proton (H) from his to the amino group formed by cleavage of the peptide bond
  4. release on the amine component
    • left with acyl-enzyme
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14
Q

Stage 2 – deacylation of the ezyme

A
  1. water binding
    • takes space from amine molecule
  2. nucleophilic attack of water on the acyl-enzyme intermediate
    • His takes proton (H) from water
    • OH- attacks carbonyl C of acyl à tetrahedral intermediate
  3. collapse of the tetrahedral intermediate
    • forms carboxylic acid product
  4. release of the carboxylic acid intermediate
    • readies enzyme for another round of catalysis
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15
Q

S1 (sub) pocket

A

deep hydrophic pocket of chymotrypsin into which the long, uncharged side chains of residues can fit
• chymotrypsin prefers bonds just past residues with large, hydrophobic side chains
* the binding of an appropriate side chain into this pocket positions the adjacent peptide bond into the active site for cleavage

16
Q

The specificity of chymotrypsin depends on

A

which amino acid is directly on the amino-terminal side of the peptide bond to be cleaved

17
Q

Bond to be cleaved

A

scissile bond

18
Q

Numbering to amino side

A

Residues to the amino terminal side of the sessile bond numbered
P1 P2 P3 (subs)
corresponding sites on enzyme numbered
S1 S2 S3 (subs)

19
Q

Numbering to carboxyl side

A

Residues to the carboxyl side of the sessile bond numbered
P1’ P2’ P3’ (subs)
corresponding sites on enzyme numbered
S1’ S2’ S3’

20
Q

Chymotrypsin cleaves at peptide bonds

A

after residues with an aromatic or nonpolar side chain

21
Q

Trypsin cleaves at peptide bonds

A

after residues with long positive side chains

eg Arginine, Lysine

22
Q

Elastase cleaves at peptide bonds

A

after amino acids with small side chains

eg Alanine, Serine

23
Q

Trypsin’s active site

A
  • Aspartate 189 instead of serine

* attracts and stabilizes a positive arginine/lysine in substrate

24
Q

Elastases active site

A
  • 2 residues at top replaced with bulkier valine residues (216, 190)
  • close to mouth of pocket = only small side chains enter
25
Q

Subtilisim

A

• protease in bacteria
• catalytic triad and oxyanionic hole
BUT 1 of NH that forms oxyanion hole forms side chain of aspartate instead of polypeptide backbone
(convergent evolution)

26
Q

In addition to serine, 3 approaches to peptide bond hydrolysis

A

strategy – to generate a nucleophile that attacks the peptide carbonyl group
• cysteine proteases
• aspartyl proteases
• metalloproteases

27
Q

Cysteine proteases

A
  • Histidine activates cystein- acts as nucleophile that attacks the peptide bond
  • sulphur better nucleophle than oxygen is in serine
  • only need His and Cys = not triad
  • eg papain
28
Q

Aspartyl proteases

A

• 2 aspartic cid residues allow water to attack peptide bond
1st (deprotonated) – activates water by posing it for deprotonation
2nd (protonated) – polarizes peptide carbonyl = more susceptible to attack
• may have existed as 2 separate units (2 copies of gene for ancestral enzyme
• eg HIV and retroviruses

29
Q

Metalloproteases

A
  • active site with bound metal ion (esp Zinc)
  • activates water to act as a nucleophile to attack the peptide carbonyl group
  • eg carboxypeptidase A
30
Q

Cysteine, Aspartyl, and Metalloproteases cleave peptide bonds

A

active site with features to

  1. activate a water molecule or other nucleophile
  2. polarize the peptide carbonyl group
  3. stabilize a tetrahedral intermediate
31
Q

HIV protease

A
  • protease inhibitor
  • dimeric aspartyl protease
  • cleaves multidomain viral proteins into their active forms
32
Q

Indinavir

A
  • HIV protease inhibitor
  • blocks HIV protease from cleaving into functional units = virus not infectious
  • resembles peptide substrate of HIV protease
  • alcohol mimics tetrahedral intermediate
  • enters active site, adopts conformation that approximates symmetry of the enzyme
  • OH of alcohol interacts with 2 aspartate residues of active site
33
Q

Protease inhibitors used as drugs

A

must be specific for 1 enzyme without inhibiting other proteins in the body to prevent side effects

Basic Biochemistry (15 decks)

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