Ch 9.3

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DesLee26
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285508
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Ch 9.3
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2014-10-11 11:03:23
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Sam
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Biochem
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Test Two
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  1. ·         For bacteria to protect themselves against viral DNA, they have __ to do what?
    o   They recognize __, in their target DNA and do what?
    §  __ cleave DNA where? Other types cleave DNA at positions somewhat distant from their recognition sites
    • restriction endonuclease
    • degrade the viral DNA on its introduction into a cell

    • particular base sequences, called recognition sequences or recognition sites
    • cleave that DNA at defined positions

    • Type II restriction enzymes
    • within their recognition sequences
  2. ·         Very specific due to the fact that they must what?
    not degrade host DNA containing the recognition sequences and they must only cleave DNA molecules that contain recognition (cognate DNA) sites without cleaving DNA molecules that lack these sites
  3. ·         Restriction enzymes must cleave DNA only at __, without cleaving at other sites
    o   To keep from damaging host-cell DNA, restriction enzymes must cleave __ much more than 4000 times as efficiently as they cleave nonspecific sites
    ·         Cleavage is by __of __ from __by __
    o   A __ catalyzes the __
    §  Specifically, the bond between the 3’-oxygen atom and the phosphorus atom is broken
    • recognition sites
    • cognate DNA molecules
    • in-line displacement 
    • 3’-oxygen
    • phosphorus 
    • magnesium-activated water

    • restriction endonuclease
    • hydrolysis of the phosphodiester backbone of DNA
  4. o   The products are DNA strands with a free 3__ and a __ at the cleavage site
    §  This reaction proceeds by __ at the phosphorus atom
    ·         Two mechanisms are used:
    o   Mechanism one: __
    o   Mechanism two: __
    • ’-hyroxyl group
    • 5’-phosphoryl group
    • nucleophilic attack

    • through a covalent intermediate, employing a potent nucleophile
    • direct hydrolysis
  5. ·         In either case, each reaction takes place by __. Explain it in depth.
    in-line displacement

    The incoming nucleophile attacks the phosphorus atom, and a pentacoordinate transition state is formed, which has trigonal bipyramidal geometry centered at the phosphorus atom, with the incoming nucleophile at one apex of the two pyramids and the group that is displaced at the other apexà inversion
  6. ·         The two mechanisms differ in the __
    number of times that the displacement takes place int eh cource of the reaction
  7. o   First type: __
    §  It is then __to produce the final products; and, ultimately, __ take place at the phosphorus atomà __--> no change in __
    a nucleophile in the enzyme attacks the phosphate group to form a covalent intermediate

    • hydrolyzed 
    • two displacement reactions
    • double inversion
    • stereochemistry
  8. o   Second type: __
    §  A __ takes place at the phosphorus atomà __
    an activated water molecule attacks the phosphorus atom directly

    • single displacement reaction
    • inversion
  9. How do they determine which type it is? What was revealed?
    • ·         oxygen is replaced with sulfur and water that is coming in is enriched in 18O to allow the incoming oxygen atom to be marked. The location determines whether it is inversion or retention.
    • o   The analysis revealed that the stereochemical configuration at the phosphorus atom was inverted only once with cleavage
  10. ·         Restriction enzymes require __for catalytic activity. Their functions are determined in numerous ways:
    o   In one, crystals of EcoRV endonuclease are prepared bound to __that contain the __and grown in __. They are then soaked up. They can also be grown in a __; or, they can be __
    §  In all cases, no cleavage takes place
    • magnesium 
    • oligobucleotides 
    • enzyme’s recognition sequence 
    • absence of magnesium

    • mutated enzyme
    • replaced with other nonmetallic ions that don’t result in much catalytic activity
  11. ·         One ion binding site is occupied in all structures; and, the metal ion is coordinated to the protein through __ and __ near the __. This metal ion binds the __ that __, helping to __ and __ it in a manner similar to that for the Zn2+ ion of carbonic anhydrase
    ·         The recognition sequences for most restriction enzyme are __, which gives the __
    • two aspartate residues
    • to one phosphate group oxygen atoms
    • near the site of cleavage
    • water molecule
    • attacks the phosphorus atom
    • position and activate

    • inverted repeats
    • 3D structure a twofold rotational symmetry
  12. Structure of enzymes
    o   Dimers whose two subunits are related by twofold rotational symmetry, which facilitates the recognition of cognate DNA by the enzyme
  13. ·         An enzyme’s __ for substrates often determines __. The specificity doesn’t come in the __, but the __. The answer lies in a unique set of interactions between the enzyme and a cognate DNA sequence
    o   Within the 5’-GATATC-3’ sequence, the G and A bases at the 5’ end of each strand and their Watson-Crick partners directly do what?
    • binding affinity
    • specificity
    • type of DNA bound
    • sequence that gets cleaved

    contact the enzyme by hydrogen bonding with residues that are located in two loops, one projecting from the surface of each enzyme subunit
  14. §  The most striking feature of this complex is the __, which is __in the center. The central two __ play a key role in producing the __. They do not make contact with the enzyme but appear to be required because of their __
    ·         5’-TA-3’ sequences are known to be among the most easily __. The distortion of the DNA at this site has severe effects on the __
    • distortion of the DNA
    • kinked 
    • TA base pairs in the recognition sequence
    • kink
    • ease of distortion
    • deformed base pairs
    • specificity of enzyme action
  15. o   Specificity can be determined by __
    §  Although EcoRV endonuclease (for example) binds to both __ and __ with equal affinity, the structures of complexes formed are different:
    ·         The noncognate DNA conformation is not __--> no __ is positioned sufficiently close to the __ to __; and, the nonspecific complexes don’t __ and the __. The distortion of substrate and subsequent __ account for the __ of more than 1,000,000-fold that is observed for EcoRV endonuclease despite little preference at the level of substrate binding
    • enzyme’s binding affinity for substrates
    • cognate and noncognate DNA
    • distortedà 
    • phosphate
    • active-site aspartate residues
    • complete a magnesium ion binding site
    • bind the magnesium ion
    • catalytic apparatus is not assembled
    • binding of the magnesium ion
    • catalytic specificity
  16. §  Thus, __ is needed to attain __. When binding to enzymes, the DNA is distorted in such a way that __, increasing the __.

    • binding energy
    • catalytic specificity
    • additional contacts are made between the enzyme and substrate
    • binding energy
  17. But, this increase is canceled by the __. Thus, for EcoRV endonuclease, there is little difference in __ for cognate and nonspecific DNA fragments. However, the distortion in the cognate complex dramatically affects catalysis by completing the __
    • energetic cost of distorting the DNA from its relaxed conformation
    • binding affinity
    • magnesium ion binding site
  18. o   This illustrates how enzymes can utilize available __ to __ and poise them for __. Interactions that take place within the distorted substrate complex __, leading to __
    • binding energy
    • deform substrates
    • chemical transformation
    • stabilize the transition state
    • DNA hydrolysis
  19. §  Distortion in the DNA explains how __blocks catalysis and protects host-cell DNA. Explain.
    methylation 

    When a methyl group is added to the amino group of the adenine nucleotide at the 5' end of the recognition sequence by enzymes called methylases, the methyl group's presence precludes the formation of a hydroen bond between the amio group and the side chain carbonyl group of the asparagine, which is closely linked to the other amino acids that form specfic contacts with the DNA. The absence of the hydrogen bond disrupts other interactions between the enzyme and the DNA substrate, and the distortion necessary for cleavage will not take place
  20. ·         An endonuclease will not cleave DNA if its __ is __. These pairs of enzymes are __
    o   The distortion in the DNA explains how methylation does what?
    • recognition sequence is methylated
    • restriction-modification systems
    • blocks catalysis and protects host-cell DNA.
  21. ·         Restriction enzymes are not significantly similar. But, the 3D structures revealed the presence of a core structure conserved in the different enzymes, which includes __--> evolutionarily related
    o   __ may have led bacteria to acquire the similar genes; so, they may have obtained the gene for these restriction endonucleases from a common source more recently than the time of their __
    • beta strands that contain the aspartate residues forming the magnesium ion-binding sites
    • Horizontal gene transfer
    • evolutionary divergence

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