More enzyme mechanisms

Question 1

Outline the mechanism of restriction endonucleases

Answer 1

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

Question 2

Describe EcoRI and EcoRV

Answer 2

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

Question 3

Why are Mg and Mn used in enzymes?

Answer 3

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

Question 4

Describe Mva1

Answer 4

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

Question 5

How can restriction endonuclease activity be abolished?

Answer 5

Replacing phosphate with a phosphorothioate/methylphosphonate

Question 6

Describe a two metal ion mechanism:

Answer 6

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

Question 7

Describe the HNH domain of Cas9

Answer 7

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

Question 8

Describe the E7 colicin structure

Answer 8

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

Question 9

What are the active site residues of HIV protease inhibitors?

Answer 9

Asp25A and Asp25B

Question 10

Describe HIV protease mechanism

Answer 10

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.

Question 11

What does peptide deformylase do?

Answer 11

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

Question 12

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

Answer 12

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

Question 13

What sort of inhibitors target PDF C-terminus?

Answer 13

Allosteric inhibitors

Question 14

What parameters are considered for binding affinity of inhibitors?

Answer 14

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

Question 15

How is reaction rate calculated?

Answer 15

Kcat x [ES]

Question 16

How can a K140Q mutation in isocitrate dehydrogenase affect biosynthetic pathways

Answer 16

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

Question 17

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

Answer 17

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