Chpt 10 b Flashcards
Reversible Covalent Modification
Used to turn proteins on or off
Phosphorylation
- dephosphorylation
- acetylation
Phosphorylation
-reversible attachment of phosphate group (usually gamma phosphate) to hydroxyl of R group of S, T, Y
Catalyzed by Protein Kinase
- Enzyme that transfer a phosphate from from ATP to R group hydroxyl on S,T, Y of Protein
- Serine/threonine protein kinases
- Tyroseine kinases
Phosphorylation works well to regulate activity because:
- Phosphate’s two negative charges disrupt electrostatic interaction
- Phosphate bonds are directional
- Large free energy
- Rapid rate (less than sec)
- amplified effect
- Use of ATP ties phosphorylation to energy charge of cell
Dephosphorylation
-Removal of phosphate from phosphorylated protein
Catalyzed by Protein Phosphatase
- enzymes that removes a phosphate from phosphorylated protein
- Serine/threonine protein phosphatase
- tyrosine protein phosphatase
Phosphorylation/Dephosphorylation
At physiological conditions
- irreversible
- rate of uncatalyzed reaction is negligible
Kinases/Phosphatases maybe:
1) Dedicated Kinases/Phosphatases
- phosphylates/dephosphorylates specific target
2) multifunctional Kinases/Phosphatases
- phosphylates/dephosphorylates multiple targets
Acetylation
attachment of an acetate group to the R group of Lysine
-Histones are acetylated and deaceltylated
Cyclic AMP (cAMP)
Phosphate connects 3’ carbon to 5’ carbon
- second messenger
- Example: amplification of fight or flight signal from hormone epinephrine (adrenaline)
Protein Kinase A (PKA)
Heterotetramer of two subunits (R2C2)
1) Catalytic subunit:
- phosphorylates large proteins (protein kinase activity) when freed by R subunit
2) Regulatory Subunit
- each R subunit contains two binding site for cAMP
Numerous Isozymes
PKA participates n “fight or flight” response
- Hormone epinephrine (adrenaline) binds membrane receptor
- stimulates synthesis of cAMP (second messenger) by adenylate cyclase
- Two cAMP bind to regulatory subunit of PKA
- catalytic subunit is activated and functions as kinase
Pseudosubstrate binds to active site of the C subunit
X-ray Crystal structure of PKA
Inhibitor and ATP-Mg2+ bind to active site of PKA catalytic subunit
Two lobes:
- smaller lobe binds ATP-Mg2+
- Large lobe binds protein and contains catalytic residues
Binding of substrate causes a conformational change in PKA resulting in the two lobes moving closer to each other
Proteolytic activation
-def
an enzyme is activated by cleaving peptide bond by proteolysis
Zymogens
Zymogen or proenzyme
- inactive precursor of enzyme
- often activated by proteolysis
use secretory pathway:
1) rER-proteins (zymogens) synthesize
2) Transport vesicles (COP II)
3) Golgi- Clathrin coated
4) Stored vesicles (granules)
- stored until nerve impulse or hormonal signal is received
5) Secreted
Processes using zymogens
Digestive enzymes Blood Clotting Protein hormones -preproinsulin-proinsulin-insulin Collagen Development Apoptosis
Synthesis and activation of Chymotrypsin
Chymotrypsin synthesized as 245 amino acid zymogens called chymotrypsinogen in acinar cells of pancreas
- stored in membrane bound vesicles (zymogen granules) until needed
- released into duct of duodenum
Proteolysis of peptide bond between amino acid 15 and 16 causes conformational change
- ile16 turns inward and forms ionic bond to Asp
- Met moves to surface
- Residues 187 to 193 become extended, which begins creating of hydrophobic substrate binding cleft
- oxyanion hole is incomplete
Hydrolysis of single peptide bond causes discrete conformational change
Enteropeptidase
activates trypsin by hydrolyzing lys-6 ole-7 peptide in trypsinogen
Trypsin
- activates more trypsin and other zymogens
- hydrolyzes proteins on c terming side following long, positively charge R groups (R, K)
Elastase
Cleaves peptide on C terminus of small chains (A,S)
Carboxypeptidase
cleaves c terminal amino acids from protein/peptides
Lipase
digests lipids
Trypsin inhibitor
Inhibits trypsin in pancreas and pancreatic duct:
- binds tightly to trypsin’s active site
- survives denaturing agents: 8M urea and 6M guanidine hydrochloride
Trypsin inhibitor is an effective substrate analog
- Lys of trypsin inhibitor interacts with asp of trypsin
- slow reaction rate
Blood Clotting Pathway
Intrinsic pathway
-triggered by exposure of anionic surfaces on rupture of endothelial lining of blood vessels
Extrinsic pathway
-trauma exposes tissue factor (TF)an integral membrane glycoprotein
Final common pathway
Fibrinogen (I) -> Fibrin (Ia)
6 subunits
-2 each (A, a, B, b, Y)
Thrombin hydrolyzes four arg-gly peptide bonds to activate fibrinogen to fibrin monomer (aBy)2
-four fibrinopeptides removed: two from A chain and B chain each
Fibrin Monomoer assembles into fibrous arrays called protofibril
Protofibril interact with each other to form soft clot
Soft clot is stabilized by formation of amide bonds between side chains of lys and gln in different monomers
catalyzed by transglutaminase (Factor XIIIa)
Prothrombin (II) -> Thrombin (IIa)
Prothrombin 4 domains
- Gla domain=y-carboxyglutamate rich=modified amino acid with two carboxylic acids
- two Kring domain=resemble pastry
- serine protease domain
Xa activates prothrombin cleavage by making 2 proteolytic cleavages:
- Arg 274- thr 275
- Arg 323 -ile 324
y-carboxyglutamate binds Ca2+
-the enzyme that adds the second carboxylic acid requires vitamin K
Ca2+ causes prothrombin to attach to phospholipid membranes derived from blood platelets after injury
Vit K
required for synthesis of prothrombin
Vit K antagonists:
1) dicoumarol
- compound found in spoiled sweet clover
- fatal hemorrhagic disease in cattle
- cattle synthesize abnormal prothrombin that does not bind Ca2+
2) Warfarin
- Clinical Anticoagulant
- Rat poison
Hemophilia A
Sex linked recessive
Defective factor VIII
-intrinsic pathway missing/reduced
Normal Factor VIII
-stimulates Factor IXa (serine protease) to activate factor X ( a protease)
Current treatment for hemophilia A involves transfusion with Factor VIII, produced using recombinant DNA technology
Break down of clots
Clots dissolve when integrity of damaged area is restored
Plasmin
- activated by TPA (tissue-type plasminogen activator) cleaving plasminogen
- serine protease that hydrolyzes peptide bonds in fibrin clots
TPA
- structure related to Prothrombin
- used clinically to break up clots during MI
