Ch 9.1

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Ch 9.1
2014-10-11 09:50:26
Test Two
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  1. ·         __ is important. What must happen to Proteins that have done their job?
    o   Proteases cleave proteins by a __. Though thermodynamically favored, they are __ in the absence of a catalyst
    Protein turnover

    must be degraded so the amino acids can be recycled; and proteins ingested must be degraded for absorption

    hydrolysis reaction

    very slow
  2. ·         The chemical bonding in peptide bonds is responsible for their __. Specifically, the resonance structure that accounts for the planarity of a peptide bond also makes such bonds __.
    o   The carbon-nitrogen bond is strengthened by its __. Furthermore, the carbonyl carbon atom is __ and __ than are the carbonyl carbon atoms in more reactive compounds. 

    §  As a result, to promote peptide-bond cleavage, an enzyme must __ at a normally __
    • kinetic stability
    • resistant to hydrolysis

    • double bond character
    • less electrophilic
    • less susceptible to nucleophilic attack

    • facilitate nucleophilic attack
    • unreactive carbonyl group
  3. ·         __cleaves peptide bonds selectively on the __; and, it uses __ to do so

    • Chymotrypsin 
    • carboxyl-terminal side of the large hydrophobic amino acids such as Trp, Tyr, Phe, and Met
    • covalent catalysis
  4. Explain how chymotrypsin reacts?

    What does it possess?
    o   A powerful nucleophile attacks the unreactive carbonyl carbon atom of the substrate and the nucleophile becomes covalently attached to the substrate in the course of catalysis

    o   Chymotrypsin possesses a reactive serine residue, which plays a central role in the catalytic mechanism of chymotrypsin
  5. ·         Chymotrypsin action proceeds in two steps linked by a __; and, the kinetics of enzyme action can be monitored by having the enzyme act on a __ that forms a colored product
    o   For chymotrypsin, such a __substrate is ___. This substrate is an __rather than an __, but many proteases will also hydrolyze __. One of the products formed by chymotrypsin’s cleavage of this substrate is __, which has a yellow color. 
    • covalently bound intermediate
    • substrate analog

    • chromogenic 
    • N-acetyl-L-phenylalanine p-nitrophenyl ester
    • ester 
    • amide
    • esters
    • p-nitrophenolate
  6. o   Under steady-state conditions, the cleavage of this substrate obeys __.
    §  The initial phase of the reaction s examined by using the __, which mixes enzyme and substrates and monitors
    ·         This method results in a ra__, followed by __ ast he reaction reaches the __
    • MM kinetics
    • stopped-flow method
    • pid burst of colored product
    • slower formation
    • steady state
  7. o   These results suggest that hydrolysis proceeds in two phases. What are they?
    • §  In the first reaction cycle that takes place immediately after mixing, only the first phase must take place before the colored product is released.
    • §  In subsequent reaction cycles, both phases must take place
  8. o   The two phases are explained by the formation of a __
    §  First, the __ of the substrate becomes __ to the enzyme as __ is released
    ·         The enzyme-acyl group complex is called the __
    covalently bound enzyme-substrate intermediate

    • acyl group
    • covalently attached
    • p-nitrophenolate

    acyl-enzyme intermediate
  9. §  Second, the acyl-enzyme intermediate is __to __ and __
    ·         Thus, one molecule of __ is produced rapidly from each enzyme molecule as the __ is formed.
    ·         However, it takes longer for the enzyme to be “__” by the __, and both phases are required for __ 
    • hydrolyzed 
    • release the carboxylic acid component of the substrate and regenerate the free enzyme

    • p-nitrophenolate
    • acyl-enzyme intermediate

    • reset
    • hydrolysis of the acyl-enzyme intermediate
    • enzyme turnover
  10. ·         __is part of the __ that also includes __ and __
    • Serine 
    • catalytic triad
    • histidine and aspartate
  11. Explain the structure of chymotrypsin
    o   Chymotrypsin structure: roughly spherical and comprises three polypeptide chains, linked by disulfide bonds. It is synthesized as a single polypeptide, chymotrypsinogen, which is activated by the proteolytic cleavage of the polypeptide to yield the three chains
  12. Active site of chymotrypsin
    §  The active site lies in a cleft on the surface of the enzyme. In it, the side chain of serine is hydrogen bonded to the imidazole ring of histidine. The –NH group of this imidazole ring is, in turn, hydrogen bonded to the carboxylate group of aspartate. 
  13. How does the catalytic triad work...summary wise? Steps one and two?
    • o   The histidine residue serves to position the serine side chain and to polarize its hydroxyl group so that it is poised for deprotonation.
    • o   In the presence of the substrate, the histidine residue accepts the proton from the serine hydroxyl group, acting as a general base catalyst. 
  14. How does the catalytic triad work...summary wise? Steps three and four?
    • o   The withdrawal of the proton from the hydroxyl group generates an alkoxide ion, which is a much more powerful nucleophile than is an alcohol
    • o   The aspartate residue helps orient the histidine residue and make it a better proton acceptor through hydrogen bonding and electrostatic effects
  15. ·         The mechanism of the catalytic triad:
    The reaction begins with __
    o   the oxygen atom of the side chain of serine making a nucleophilic attack on the carbonyl carbon atom of the target peptide bond (2)à four atoms are now bonded to the carbonyl carbon, arranged as a tetrahedron, instead of three atoms in a planar arrangement
  16. §  This unstable tetrahedral intermediate bears a __ on the oxygen atom derived from the __. This charge is stabilized by interactions with __
    • formal negative charge
    • carbonyl group
    • NH groups from the protein in a site termed the oxyanion hole.
  17. o   These interactions help __ that precedes the formation of the __
    §  Ths is faciliateed by the __ being held by the __ to the __ formed by __.
    o   The __ (4), completing the first stage of the __ 
    • stabilize the transition state
    • tetrahedral intermediate, which collapses to generate the acyl-enzyme (3)

    • transfer of the proton
    • positively charged histidine residue to the amino group formed by cleavage of the peptide bond

    amine component is now free to depart form the enzyme

    hydrolytic reaction—acylation of the enzyme
  18. o   The next step—__—begins when a __ (5)
    §  The __is now hydrolyzed by a process that essentially repeats steps 2 through 4
    ·         Now acting as a __, __draws a proton away from the water molecule. 
    • deacylation
    • water molecule takes the place occupied earlier by the amino component of the substrate

    • ester group of the acyl-enzyme 
    • general acid catalyst
    • histidine
  19. o   The resulting __ attacks the __, forming a __ (6)
    o   This structure breaks down to form the __ (7)
    o   Finally, the release of the __(8) readies the enzyme for __
    • OH- ion
    • carbonyl carbon atom of the acyl group
    • tetrahedral intermediate

    • carboxylic acid product
    • carboxylic acid product 
    • another round of catalysis
  20. ·         This process is characteristic of __except for areas farther from the large, hydrophobic residues
    ·         Examining the 3D structure of chymotrypsin with substrate analogs and enzyme inhibitors revealed the presence of a __, into which the __ of residues such as Phe and Trp can fit
    • chymotrypsin 
    • deep hydrophobic pocket, called the S1 pocket
    • long, uncharged side chains
  21. o   The binding of an appropriate __into this pocket positions the adjacent __ into the __ for cleavage
    §  The specificity of chymotrypsin depends almost entirely on __
    §  Other enzymes that have different specificities have different pockets 
    • side chain 
    • peptide bond
    • active site

    which amino acid is directly on the amino-terminal side of the peptide bond to be cleaved
  22. ·         Catalytic triads are found in other hydrolytic enzymes
    o   Some homologs of chymotrypsin have the triad; and the structure is similar. Examples are __ and __
    o   Despite their similar structure, they differ greatly in substrate specificity
    trypsin and elastase
  23. Chymotrypsin
    §  Chymotrypsin: cleaves at the peptide bond after residues with an aromatic or long nonpolar side chain
  24. Trypsin 
    cleaves at the peptide bond after residues with long, ppstiively charged side chains—like arginine and lysine
  25. Elastate 
    cleaves at the peptide bond after amino acids with small side chains—like alanine and serine
  26. o   The different specificities are due to small structural differences

    • §  In trypsin, an aspartate residue is present at the bottom of the S1 pocket in place of a serine residue in chymotrypsin
    • ·         the aspartate attracts and stabilizes a positively charged arginine or lysine residue in the substrate
  27. o   The different specificities are due to small structural differences

    §  in elastase, two residues at the top of the pocket in chymotrypsin and trypsin are replaced by much bulkier valine residues, which close off the mouth of the pocket so that only small side chains can enter
  28. ·         Other chymotrypsin family members are proteins involved in __ and __
    ·         Other enzymes that are not homologs of chymotrypsin have been found to contain very similar active sites, which is a consequence of __
    • blood clotting 
    • tumor marker prostate-specific antigens
    • convergent evolution
  29. ·         Other proteases have been discovered that contain an __ that is activated not by a __ but by a primary amino group from the side chain of _or by the __ of the polypeptide chain
    • active-site serine or threonine residue
    • histidine-aspartate pair
    • lysine 
    • N-terminal amino group
  30. ·         Testing for the catalytic triad?    Results?
    • o   Test the contribution of individual amino acid residues to the catalytic power of a protease by using site-directed mutagenesis: covert one of the residues within the triad to alanine to examine its effectivity
    • §  Results:
    • ·         Serineà alanine: kcat fell to less than one-millionth of its value for the wild-type enzyme; Km was unchanged
    • ·         Histidineà alanine: affected in a similar way
    • ·         Aspartateà alanine: affected but to a lesser degree
    • ·         All threeà same effect as converting serine or histidine
  31. §  Prove that the __ and, particularly, the __ act together to generate a __of sufficient power to attack the __ of a peptide bond
    §  Despite the reduction in their catalytic power, the mutated enzymes still hydrolyze peptides a thousand times as fast as buffer at pH 8.6
    • catalytic triad
    • serine-histidine pair
    • nucleophile 
    • carbonyl carbon atom
  32. o   __ also allowed probing of the importance of the oxyanion hole for catalysis
    §  The mutation of __ to __ elimitated the side chain NH group from the __ of subtilisin, which reduced the value of __to 0.2% of its wild-type value but increased value of __by only a factor of two. These observations demonstrate that the __ residue plays a significant role in stabilizing the __ and the transition state leading to it
    • Site directed mutagenesis
    • asparagine to glycine
    • oxyanion hole
    • kcat 
    • Km 
    • NH group of the asparagine
    • tetrahedral intermediate
  33. ·         Classes of proteins have also been discovered that employ three alternative approaches to peptide bond hydrolysis: __, __, and __
    o   In each case, the strategy is to __ 
    • cysteine proeases, aspartyl proteases, and metalloproteases
    • generate a nucleophile that attacks the peptide carbonyl group
  34. Cysteine proteases
    • ·         cysteine residue, activated by a histidine residue, plays the role of the nucleophile that attacks the peptide bond in a manner similar to that of serine
    • o   Because sulfur is a better nucleophile than is the oxygen atom in serine, cysteine proteases appear to require only this histidine residue in addition to cysteine and not the full catalytic triad
  35. Aspartyl proteases
    • ·         the central feature of the active sites is a pair of aspartic acid residues that act together to allow a water molecule to attack the peptide bond
    • o   One aspartic acid residue (in its deprotonated form) activates the attacking water molecule by poising it for deprotonation. The other aspartic acid resuie (in its protonated form) polarizes the peptide carbonyl group so that it is more susceptible to attack 

    §  Members of this class are renin and pepsin, which possess twofold symmetry

  36. Metalloproteases
    ·         constitute the final major class of peptide-cleaving enzymes. The active site contains a bound metal ion, almost always zinc, that activates a water molecule to act as a nucleophile to attack the peptide carbonyl group. 
  37. ·         In all three classes of enzymes, the active site includes features that act to __(3)__
    1) activate a water molecule or another nucleophile, 2) polarize the peptide carbonyl group, and 3) stabilize a tetrahedral intermediate
  38. ·         Several important drugs are protease inhibitors

    Ex: Indinavir (Crixivan)
    resembles the peptide substrate of the HIV protease. It is constructed around an alcohol that mimics the tetrahedral intermediate: other groups are present to bind into the S2, S1, S1', and S2' recognition sites on the enzyme. 

    Indinavir adopts the conformation that approximates the two-fold symmetry of the enzyme
  39. §  The active site of HIV protease is covered by __ that fold down on top of the bound inhibitor. The OH group of the central alcohol interactis with the __. In addition, two __ are hydrogen bonded to a water molecule which, in turn, is hydrogen bonded to a __ group in each of the flaps.
    o   __ used as drugs must be specific for one enzyme without inhibiting other proteins within the body to prevent side effects
    two flexible flaps

    two aspartate residues of the active site

    carbonyl groups of the inhibitor

    peptide NH

    Protease inhibitors