NEED TO KNOW

Question 1

Nucleoside Monophosphate Kinases

Answer 1

Nucleoside Monophosphate Kinase
Adenylate Kinase
Function
-Transfers a gamma phosphate from a nucleoside triphosphate to a nucleoside monophosphate producing a nucleoside disphosphate

Divalent cations (Mg2+, Mn2+, etc) required for enzymes using nucleotides as substrates
**not bound to enzyme active site, but bound to substrate (nucleotide)
-Substrate= ATP-Mg2+
Forms 6 coordination bonds (octahedral arrangement)
-two to oxygen
-4 to water
-bind to various oxygen in various combinations
-Numerous stereochemical orientations
-Increases interaction with enzyme=increased binding E

Xray crystal structures of enzymes are homologues

• Beta sheet surrounded by alpha helixes
• P-Loop

P-loop in adenylate Kinase
Phosphate Binding Site
-conserved Motife GXXXXGK binds ATP via Phosphate groups
-common to many nucleotide binding enzymes

P Loop mech:Binds to ATP-Mg2+

• Aspartic acid of adenylate kinase binds to ATP-Mg2+ complex through H-Bond to water, which induces a conformation change in adenylate Kinase (Induced Fit model)
• P loop closes over phosphates of ATP, especially B phosphate, and Gamma phosphate is now aligned next to NMP binding site
• Binding occurs at NMP binding site and causes additional conformational changes
• Catalysis by approximation

Question 2

Restriction Endonucleases

Answer 2

Endonuclease Type II
Function
-cleave specific DNA base sequence (recognition site)
-Catalyzes Hydrolysis of Phosphodiester bonds
a)produce 5’ PO4 and 3’ OH
b) Mg2+ activates water, creating a nucleophile which attacks the phosphorus of the DNA

Found in Bacteria and Archaea

• require Mg2+ or other divalent cations
  a) Mg2+ is bound by: 2 Asp or EcoR V, Phosphoryl Oxygen of DNA, and Water
• 2 mechanisms
  a) Both use pentacoordinated Intermediates
  b) differ in number of displacements

Transition state

• Bipyramid Geometry-nucleophile attached to one apex; LG attached to the other apex
• Inverted Stereochemistry

Mech 1: 2 steps

1) formation of covalent intermediate
2) Hydrolysis to final product
- 2 inversions of stereochemistry
- RETAIN orientation of phosphorus

Mech 2: Direct Hydrolysis

• Single Inversion of stereochemistry
• Inverted configuration of Phosphorus

Experiment do differentiate between 2: Hard to determine orientation of Phosphorus so use Protocol
-Phosphorothioate Labeled DNA
-Water with O18
-determine by the orientation of water’s O18 relative to S
Results- Mech 2, because the orientation of water was inverted

Question 3

EcoR V

Answer 3

EcoR V=Recognition Site 5’GATATC 3’

• cleaves methylated DNA
• protects-> methylation of 5’ adenine prevents H-bonding with Asn thus disrupting interaction between DNA and Enzyme

Interaction with Cognate DNA:
G:C BP H-bond with EcoR V
-backbone O and H-N of Gly (Diff Gly residues)
-backbone H-N of Asn
A:T BP H-bond with EcoR V
-backbone O and H-N of Asn (same Asn residue)
-r group Oxygen of Thr

X-ray structure of EcoR V
-cannot be determined due to cleaving of cognate DNA:Mg2+

Recognition Site Produces 2 fold rotational symmetry

• inverted repeats
• Restriction endonuclease binds equally with cognate DNA and non cognate DNA

Cognate DNA recognition site DISTORTS and produces additional interactions with EcoR V

• Free E (binding E) produces additional interactions with EcoR V
• Middle 5’ TA 3’ distorts and brings phosphate of DNA cleavage site into proximity of Mg binding site in active site of EcoR V. Mg binds completing catalytic apparatus:

Noncognate DNA Does NOT distort

• lack of distortion (in non cognate DNA) does not allow binding of magnesium, so catalytic apparatus not assembled
• lack of Mg2+, EcoR V binds equally well with cognate DNA and Noncognate DNA

Question 4

Break Down of Clots

Answer 4

Clots dissolve when integrity of damaged are resolved

Plasmin

• activated by TPA cleaving plasminogen
• serine protease that hydrolyzes peptide bonds found in fibrin

TPA
-used clinically to break up clots during MI

Question 5

Common pathway

Answer 5

1) Xa activates prothrombin by making two proteolytic cleaves
2) Y-carboxyglutamate binds Ca2+ and causes Prothrombin to attach to phospholipid membrane and is derived from blood after injury
3) Thrombin hydrolyzes four arg-gly peptide bonds to activate fibrinogen to Fibrin monomer
- removes four fibrinopeptides
4) Fibrin monomer assembles into fibrous array called protofibril
5) Protofibrils interact with each other to form a soft clot
6) Soft clot is stabilized by formation of amide bond between chains of lys and gln in different monomers
7) catalyzed by transglutaminase (Factor XIIIa)

Question 6

Vit K

Answer 6

Required for synthesis of prothrombin

Vit K antagonists

1) Dicoumarol
- found in spoiled sweet clover
- fatal hemmorhagic disease in cattle
- cattle synthesize abnormal prothrombin that does not bind to Ca2+

Warfarin

• clinical anticoagulant(blood thiner)
• Rat poison