More enzyme mechanisms Flashcards
Outline the mechanism of restriction endonucleases
- Land on DNA
- Linear diffusion
- Find target sequence and distort DNA
- Coupling and catalysis (metal ion has a role)
- Product release
Describe EcoRI and EcoRV
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
Why are Mg and Mn used in enzymes?
They have a well defined coordination shell that can position water
High charge density
Describe Mva1
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
How can restriction endonuclease activity be abolished?
Replacing phosphate with a phosphorothioate/methylphosphonate
Describe a two metal ion mechanism:
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
Describe the HNH domain of Cas9
His and Asp residues which establish an attacking nucleophile
Mg2+ stabilises the transition state, maybe even Ni2+, Co2+, Zn2+ and Ca2+
Describe the E7 colicin structure
may use Zn2+ or Mg2+
Conserved His and Cys residues bind Zn2+ for a structural role
What are the active site residues of HIV protease inhibitors?
Asp25A and Asp25B
Describe HIV protease mechanism
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.
What does peptide deformylase do?
Deformylates N-formyl methionine (fMet) which is the first amino acid encoded by bacteria
What is the function of the conserved PDF C-terminal?
Allows the enzyme to bind to it as a recognition site.
What sort of inhibitors target PDF C-terminus?
Allosteric inhibitors
What parameters are considered for binding affinity of inhibitors?
Kcat and Km when I and no I is in the site
How is reaction rate calculated?
Kcat x [ES]