Ch 10 Lecture 1

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Ch 10 Lecture 1
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2014-10-18 11:55:56
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Test three
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  1. Explain that convoluted maze connect the dot structure?
    dots represent intersections

    represents 500 or so reactions of metabolic pathways

    pathways are enzymes
  2. To live, organisms need __ .
    five hundred reactions
  3. How many enzymes are active at any given moment?
    not all of them because we need regulation so that reactions that don't need to happen aren't taking place
  4. What are the four regulatory strategies?
    allosteric control

    isozymes

    reversible covalent modification

    proteolytic activation
  5. allosteric control
    enzyme present but in wrong conformation; interaction of enzyme with some other molecule, causing a change in shape so enzymes becomes active to inactive
  6. Isozymes
    evolutionarily speaking, some genes of enzymes have replicated many times--> multiple copies of genes within a genome

    all are subject to mutation in different places, times, and rates

    enzymes begin to diverge

    They catalyze the same reaction, but use different specificities such as rates
  7. What do isozymes allow?
    they give cells ability to pick and choose the isozyme that functions best in the physiological environment

    cells have a choice of which enzyme
  8. Reversible covalent modification and proteolytic activation

    - similarities
    • produces enzymes in an inactive (proenzyme) form
    • enzymes synthesize but not in correct conformation to be active. Can be active in one or two ways, which is the reversible covalent mod and proteolytic enzymes
  9. Reversible covalent modification
    mostly through phosphorylation of serine, threonine, and tyrosine

    family of kinases that phosphorylate serine and threonine. They pop off hydrogen and phosphate, altering side chain--> conformational change in enzymes, activating it

    since it is reversible, another enzyme must take phosphate off (phosphatase- removes phosphate)
  10. What are the advantages and disadvantages of reversible covalent modification?
    already produced and ready to go (switches on and off)

    makes enzymes recyclable (no need to remake enzyme); saves energy
  11. Reversible covalent modification is __.
    the most common way of activating enzyme inside of cell where ATP is readily available.
  12. Proteolytic activation
    enzyme is present in proenzyme form, but, instead of adding something, a pep bond is cleaved, activating the enzyme

    separate protease is required

    happens outside of cells; one time deal; one protein is activated, you can't deactivate it.
  13. Why can't you deactivate an enzyme after activating it?
    its outside of the cell, you can't find it

    you don't want random proteases to roam around inside cell
  14. ATCase
    allosteric protein; first step in a pathway leading to production of nucleotide CTP; activated when we need CTP
  15. What are the specificities that occur in the reaction that ATCase catalyzes?
    • two subunits
    • 1) amino acid aspartate
    • 2) carbamoyl phosphate

    both can be used outside this reaction as well
  16. What is CTP's role in the reaction?
    it is a regulation of ATCase

    • if there is enough CTP in the syste, the end product, CTP, inhibits ATCase to stop production
    • Due to this, ATCase is the commiteted step. Once it catalyzes that reaction, it goes on to produce it. If not needed, hydrogen needs to be inhibited at this reaction.
  17. CTP is not what?
    is not the product of the reaction. It is the product of the pathway that has several reactions.

    CTP production is a multistep reaction pathway
  18. Explain ATCase
    • has T and R state; has two catalytic triads
    • self-interaction in R state versus other interactions in the T state

  19. Explain this
    With every substrate constant, we vary the concentration of inhibitor. The more inhibitor that is added, the less the reaction proceeds, showing that CTP inhibits ATCase
  20. Explain this.
    No CTP is present. In a cuvette, buffer held constand. We change [Aspartate]. 

    ATCase is an allosteric enzyme. 

    This graph shows the rate increases as the amount of substrate increases, eventually leveling off
  21. Explain the structure and format of the enzyme.
    • it has multiple subunits being held by S-S- bonds
    • ``
  22. What experiment did they do to determine ATCase structure?
    reagent broke the SS bonds and bonds to cysteine residues. They took them and ran it through cesium chloride. 

    Large centrifuge tube with layer of proteins on top. Let it spin overnight and at a higher speed. Different proteins willm igrate down and rest. Larger moves further down. Wherever a band forms shows what the mass is

    They then assayed it
  23. Explain the assay.
    12 subunits to begin with in the very begining before breaking it down

    after breaking a bond, you get 2 peaks--> one representing a dimer (r) and another representing a trimer (C)

    It has three subnits

    Different peaks corerspond to two different types of subunits
  24. What do the regulatory dimers do?
    regulate activity o eznymes and come in pairs
  25. How is it arranged?
    the trimers are held together by the dimers
  26. The reaction that occurs doesn't happen rapidly. What does it involve?
    • a reaction intermediate
    • it is not quick, but it is quick enough to prevent us from freezing
  27. How did scientists discover and study the reaction intermediate?
    to study, scientists creted an intermediate analog to study it. It can't have its phosphate group removed. Gets into active site and freezes ATCase in its active conformation--> X ray crystallography revealed structure when active versus inactive
  28. Each subunit has an __. __ and __ between subunits help do what?
    active site


    • side chains and interactions
    • hold substrate in place
  29. How to get T to switch to R.
    we have purified ATCase in tense state in cuvette--> add PALA--> binds to each subunit (6 active sites per ATCase= 6 reactions at one time)--> binds to active site--> T switches to R--> changes
  30. What are the changes from T to R?
    space in middle gets longer. 12 angstrom separation

    10 degree rotation relative to each other vertically

    15 degree rotation horizontally
  31. Inactive enzyme: T state
    binds to regulatory subunits to prevent a conformational change

    CTP helps maintain the T state

    the subunits overlap and touch in the T state
  32. Active enzyme: R state
    pull apart and rotate
  33. Explain the relationship between active sites.
    changes in one catalytic subunit, which is made of two lobes with hinge in the middle

    • T state= side chains too far apart
    • R state= movement of the two lobes; bring in closer proximity--> interaction
  34. What allows the T state to exist?
    interactions across catalytic trimers allow it to happen

    Specific amino acid side chains form ionic bonds over gap= holds T state stable and holds interactions across substrate
  35. What allows the R state to exist?
    two lobes close and substrate fits in, allowing active site to be activated

    interactions become intra (among) and hold the two lobes within
  36. Explain this.
    T state corresponds to low activity; not enough to keep the cell going and making CTP

    concerted model: all six subunits switch at the same time. All are either T or all are R ; no intermediates
  37. What does CTP do?
    binds to help maintain the T state
  38. T state is __, favored by __

    R state is __, favored by __.
    • less active
    • CTP binding
    • more active
    • substrate binding
  39. Explain this.
    lower activity in the presence of CTP

    If you add ATP, it becomes more active. Cell is i a good energetic state,it can replicatei, needs nucleotides--> makes CTP

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