Ch 9A Lecture

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Ch 9A Lecture
2014-10-12 08:31:11
Test One
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  1. four specific types of enzymes
    serine proteases: break peptide bonds (serine is in active site and is responsible for it)

    carbonic anhydrases: CO2 converted to bicarbonate and vice versa

    restriction endonucleases: defense mechanism bacteria have to defend themselves against viruses/ makes cloning possible too

    myosin: ATP and energy from breaking of H bonds to make energy availale
  2. Catalytic principles
    • binding energy
    • covalent catalysis
    • general acid-base catalysis
    • metal ion catalysis
    • catalysis by approx
  3. binding energy
    • what helps us to get over the hump of Ea. The difference is between the TS and the Ea
    • Brings Ea down so that the rxn can occur
    • every bond enzyme forms with substrate makes it easier for it happen
  4. covalent catalysis
    look at the active sites taht contain nucleophiles (electron rich molecules or substrate that reacts with sites of positive charge or polarization

    covalent bond formed between enz and substrate--> TS--> releasesd as product forms

    nucleophiles help break bond by attacking substrate
  5. General Acid Base Catalysis
    • shuttling of one H+ from one molecule to another
    • Histidine is important because of its close to neutral pKa-> can shuttle protons and move them
  6. Metal Ion catalysis
    • electron poor/ + charged substance that reacts with neg. charged groups
    • a positively charged center is needed to do three things:
    • 1) stabilize charge in interaction
    • 2) can help generate a nucl
    • 3) bind to substrate and ontribute to binding energyy and lower Ea
  7. Cat by approx
    • blind date example. 
    • Enzyme facilitates the coming together of two substrates that, in its absence, would be highly unlikely to react. Once together, they react
  8. proteases
    • goal is to break bond
    • key is that the peptide bond (since it acts like a double bond) makes the target a weak electrophile less likely to undergo attack by a nucleophile
  9. Preference of chymotrypsin? 

    What do extra electrons do to the carbonyl carbon? 

    How to figure out if serine is in active site and is reactive?
    • substrates are large, hydrophobic side chains
    • extra electrons give a bit of a negative charge
    • 1) DIPF: specfic irreversible inhibitor that finds active serine residues and covalently bonds with them, making them inactive

    2) do exp. with substrate analog, looks like substarte but is not
  10. What is the example of using a substrate analog?
    N acetyl L phenylalanine p nitrophenyl ester: it enters the active site. The enzyme binds and a reaction occurs

    it is chromogenic. So, when half of the substrate is cut, it will light up
  11. How do proteases relate to MM curves?
    not a smooth parabola; it has a steep initial rxn occuring then a leveling off

    • There is a burst phase: appearance of product which forms quickly
    • steady state phase: after time, accumulation of product decreases drastically

    2 step reaction: pase is due to formation of cov bond; deacylate, bring in H2O and release
  12. explain the catalytic triad.
    histidine: usually found near the surface because of hydrophilic properties; but, in here, it is in the active site helping with the reaction

    serine: gives proton to histidine, which loses it 

    These three (ser, his, asp) might be responsible for cleavage of the peptide bond. Histidine can help because it is pos or neg. His starts out as neutral and goes positive. Because a positive charge can be harmful, aspartate is balancing it out with a negative charge.
  13. Steps 1 and two of catalytic triad peptide bond cleavage.
    1) substrate enters. Serine gets ready to brak peptide bond. Needs to become nucleophilic. So, it gives H+ to histidine. Now, the reaction is set up

    2) Proton goes over to hisitidien, which is now positive but balanced by the negative aspartate charge, preventing denaturing. Serine attacks carbonyl carbon, making the oxygen negative. But it is stabilized by the oxyanion hole.
  14. Steps 3 and 4 of catalytic triad peptide bond cleavage.
    A tetrahedral intermediate is formed, but collapses to general the acyl enzyme. This step is facilitated by a proton transfer. Serine gives Histidine a proton, which goes to the peptide bond to break it and goes to Nitrogen, allowing the amine component to be free; serine is now trapped in an acyl enzyme intermediate
  15. Steps 5 and 6 of catalytic triad peptide bond cleavage.
    • 5) water comes in and binds in the location that the amine just departed from. It goes to active site. One proton goes to Histidine.
    • 6) The remaining hydroxide is a strong nucleophile adn attacks the carbonyl carbon. The result is another tetrahedral intermediate. the negative charge of oxygen is stabilized again by the oxyanion hole
  16. Steps 7 and 8 of catalytic triad peptide bond cleavage.
    7) the tetrahedral intermediate collapses again, forming the carboxylic acid product

    8) the carboxylic acid component is released, allowing the enzyme to be available for another round
  17. The first four steps can be called the __. 
    The second four steps can be called the __.
    • burst phase
    • deacylation portion
  18. Explain the oxyanion hole
    when the tetrahedral interaction forms, oxygen is negative. So, the oxyanion hole will stabilize it
  19. Why is chymotrypsin specific to large, hydrophobic amino acids?
    because the active site is surroudned by small hydrophobic side chains, creating space to allow them to fit in
  20. What is the varying active sites of trypsin, chymotrypsn, and elastase a cause of?
    divergent evolution. A mutation converted the hydrophobic side chain to aspartic acid, making it specific for lys and arg in trypsin.
  21. Explain the binding pockets of chymotrypsin, trypsin, and elastase.
    chymotrypsin: large pocket surrounded by hydro. side chains

    trypsin: aspartic acid on the bottom, attracting positively charged lysine and arginine

    elastase: also a serine protease; has the same binding site but two small hydrophobic side chains have mutated to two larger hydropobic chains, making elastase ideal for small hydrophoibic side chains
  22. What is an example of convergent evolution and explain?
    carboxypeptidase II: looks nothing like chymotrypsin--> different protein entirely but hast eh same rxn mecahnisms as serine. Serine was such a good idea that the other enzymes modeled it
  23. What is subtilisin?
    • another serine protease
    • in lab, they mutated it hrough site directed mutagenesis. They changed each aa of the catalytic triad, one at a time, to alanine and recorded the effects
  24. What are the effects of the site directed mutagenesis?
    all three are needed to successfully run the reaction

    serine is the most important since it is the nucleophile

    histidine accepts proton

    aspartic acid is needed but the reaction can still occur without it to some extent.
  25. How can you clone the enzyme to do such a test as site directed mutagenesis?
    grab DS DNA corresponding to the gene, splice it up, put it in a plasmid, grow in bacteria, take the plasmid and alter an amino acid one at a time to a neutral amino acid that will have no effect
  26. Convergent evolution resulted in what other types of proteases?
    cysteine proteases: in active site, cysteine and histidine residue


  27. True or False: No triad is still better than an uncatalyzed reaction?
    True becuase the binding energy between sub and enz are lowering Ea, allowing the reaction to occur
  28. aspartyl proeases
    two charged aspartic acids--one charged and one uncharged

    unique because they should naturally be deprotonated. But, because of unique characteristics of microenvironment, one remains protonated

    • uncharged asp helps stabilize intermediate
    • direct hydrolysis: water is the cosubstrate; neg. charged asp helps create a nuc by taking H off H2O--> -OH
  29. Metalloproteases
    metal ion associated with catalysis; direct hydrolysis, no two steps

    • we need a basic amino acid side chain nearby
    • left with nucleophile
  30. HIV

    What can antiviral DNA do?
    • retrovirus; genome is RNA in structure
    • has integrase: which integrates virus into host genome

    proteins on the surface that bidn receptors on the cell and allow the genome to be injected

    reverse transcrptase: RNA--> DNA

    frequently attack proteases
  31. What is the mechanism of action for HIV?
    virus attaches to cell protein receptors and injects genome, which uncoats and makes RNA through reverse transcriptase--> dsDNA

    • Integrase cuts host DNA and inserts it into viral DNA and fuses the ends. The 
    • All genes are transcribed together and then translated together. One large protein gets cut into individual proteins
  32. Explain indinavir.
    it is a substrate analog that has key amino acids in cleavage of peptide bonds located in the active site. Stops the large protein from being cut up. By inhibiting proteolysis, virus doesn't spread