More enzyme mechanisms Flashcards

1
Q

Outline the mechanism of restriction endonucleases

A
  1. Land on DNA
  2. Linear diffusion
  3. Find target sequence and distort DNA
  4. Coupling and catalysis (metal ion has a role)
  5. Product release
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2
Q

Describe EcoRI and EcoRV

A

Signature PD…D/E xK motif
Utilize metal ions for catalysis
Enhance deprotonation by attacking water
Stabilize negative charges on pentavalent phosphate intermediate
Stabilises to leaving 3’ oxyanion

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3
Q

Why are Mg and Mn used in enzymes?

A

They have a well defined coordination shell that can position water
High charge density

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4
Q

Describe Mva1

A

Restriction endonuclease positions a water to attack the phosphate group
Metal group stabilises the OH group for attacking
A consistent position near scissile phosphate with a divalent metal ion

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5
Q

How can restriction endonuclease activity be abolished?

A

Replacing phosphate with a phosphorothioate/methylphosphonate

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6
Q

Describe a two metal ion mechanism:

A

MgI deprotonates and water molecule and base. Helps position the nucleophilic ion generated
MgII interacts with the leaving anionand positions a water molecule for proton donation
Ideally, these Mg ions are 4Å apart and stabilize the 2- charge on pentavalent phophorus

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7
Q

Describe the HNH domain of Cas9

A

His and Asp residues which establish an attacking nucleophile
Mg2+ stabilises the transition state, maybe even Ni2+, Co2+, Zn2+ and Ca2+

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8
Q

Describe the E7 colicin structure

A

may use Zn2+ or Mg2+
Conserved His and Cys residues bind Zn2+ for a structural role

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9
Q

What are the active site residues of HIV protease inhibitors?

A

Asp25A and Asp25B

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10
Q

Describe HIV protease mechanism

A

Asp25A and Asp25B stabilises H2O. This attacks the C=O bond on the peptide.
Asp25A then attacks the OH on the tetrahedral intermediate, the other Asp 25 is then deprotonated.

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11
Q

What does peptide deformylase do?

A

Deformylates N-formyl methionine (fMet) which is the first amino acid encoded by bacteria

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12
Q

What is the function of the conserved PDF C-terminal?

A

Allows the enzyme to bind to it as a recognition site.

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13
Q

What sort of inhibitors target PDF C-terminus?

A

Allosteric inhibitors

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14
Q

What parameters are considered for binding affinity of inhibitors?

A

Kcat and Km when I and no I is in the site

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15
Q

How is reaction rate calculated?

A

Kcat x [ES]

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16
Q

How can a K140Q mutation in isocitrate dehydrogenase affect biosynthetic pathways

A

IDH2 converts isocitrate to α-ketoglutarate. Mutant IDH2 converts this to 2-hydroxyglutarate

17
Q

What effects can build up of 2-hydroxyglutarate have on a cell?

A

Inhibits α-ketoglutarate dependent histone demethylases and methylcytosine dioxygenases
Common in acute myeloid leukaemia