Biochem: exam 4- lecture 3, ch. 10 proteolytic activation

What are the 4 basic points to know about blood clotting?

1. Blood clots are formed by a series of zymogen activations - a cascade. (requires many enzymes) 2. Very small amounts of the initial factors are required - catalytic nature of cascade leads to large amplification. 3. This ensures a rapid response to trauma; 4. Blood clotting cascade is a good illustration of regulatory strategies (enzyme regulation).

What is proteolytic activation?

1. Proteases cleaving peptide bonds to activate an enzyme 2. Many enzymes are active as soon as they are synthesized & have folded. Others are synthesized somewhere you don't want them to be active- they are activated after being transported to the appropriate place. These enzymes can be synthesized as zymogens (inactive precursors). 3. Zymogens are activated by proteolytic cleavage, which can occur outside the cell because it does NOT require ATP. 4. will only occur once in the lifetime of an enzyme (b/c it's irreversible)

What is thrombin? (3)

1. Thrombin is synthesized as prothrombin, and is activated via proteolytic cleavage. 2. Resulting thrombin is a serine protease that is homologous to trypsin. 3. Vitamin K is essential for the synthesis of prothrombin and other clotting factors.

What is trypsin? (3)

1. Trypsin is synthesized as inactive trypsinogen in the pancreas 2. Trypsin is a protease 3. Trypsin is the common activator of all the pancreatic zymogens

Trypsin is the common activator of all the pancreatic zymogens. What are some zymogens that trypsin activates?

1. chymotrpsinogen --> chymotrypsin 2. proelastase --> elastase 3. procarboxypeptidase --> carboxypeptidase 4. prolipase --> lipase 5. can even activate itself (trypsinogen --> trypsin)

X cleaves A to B. A is an inactive form of a protease while B is the active form of that protease. B cleaves C to D. C is an inactive form of a protease while D is the active form of that protease. And so on.. - Assume: One enzyme can convert 100 substrates to 100 products. - Question: if one X is activated, how many F will be produced?

100 x 100 x 100

How many enzymes in the blood clotting cascade are activated by thrombin? What are they?

4 1. Factor 7 (VII) that starts off the extrinsic pathway 2. Factor 8 (VIII) that helps activate factor 10 (X) towards end of intrinsic pathway 3. Factor 5 (V) that joins w/ factor 10 (X) to convert prothrombin (II) into thrombin at beginning of the final common pathway 4. Factor 13 (XIII) that helps fibrin monomers be joined into polymers that make up clots at the end of the final common pathway

How are blood clots dissolved?

A serine protease, tissue plasminogen activator (TPA), proteolytic activates plasminogen into plasmin. Plasmin is a serine protease that lyses fibrin clots/

Describe the final common pathway of the blood clotting cascade.

Activated factor 10 (X) amplified due to convergence of the two pathways. Factor 10 (X) works with factor 5 (V)- which is activated by thrombin- to activate prothrombin (II) into thrombin --> activates fibrinogen (I) into fibrin --> joins w/ factor 13 (activated by thrombin) to form a polymer of fibrin that creates a cross-linked fibrin clot.

What are 3 examples of proteolytic activation?

Chymotrpsin, trypsin, elastase

Why must proteolytic digestive enzymes be turned off? What is used to turn them off?

Need to be inactivated to prevent them from continuously breaking down proteins. Specific protease inhibitors are used to turn them off by binding to the active site of the proteolytic enzyme and inhibiting the substrate from entering the active site- inhibiting enzymatic activity.

What is used to inhibit trypsin?

Pancreatic trypsin inhibitor binds to the active site of trypsin, inhibiting the enzyme to prevent its over-reacting. This binding is very strong. Pancreatic trypsin inhibitor is a substrate analog (resembles the substrate molecule; has a structure complementary to the active site). Lysine 15 (+) of the inhibitor binds to the aspartate 189 (-) in trypsin active site. This interaction along w/ other interactions make the inhibitor look like a true substrate.

Why is vitamin K important? Where do we get it? What happens if we have a shortage of it?

Vitamin K is essential for the synthesis of prothrombin and other clotting factors. We cannot make it in our bodies so we must obtain it from foods- such as green vegetables. If we do not have enough vitamin K, we will lack clotting factors.

The most important inhibitor for inhibitting blood clotting is _______________.

antithrombin III

What is chymotrypsin?

digestive enzyme; serine protease that breaks peptide bonds on the COOH side of amino acids w/ bulky, hydrophobic aromatic side chains

How is trypsinogen activated?

enteropeptidase (duodenum) cleaves trypsinogen to trypsin- changing 3D structure and producing proper active site.

conversion of a zymogen via proteolysis is ______________.

irreversible

Fibrinogen activation requires _____________.

thrombin

What is the name of the inactive form of chymotrypsin? How is it activated. (5)

1. Chymotrypsin is initially formed in its zymogen form- chymotrypsinogen. Chymotrypsinogen is a single polypeptide chain w/ 245 amino acids. 2. Another protease, trypsin, is responsible for activating chymotrypsin. Trypsin cleaves a single peptide bond in chymotrypsinogen b/w the 15th [Arg] & 16th [Ile] aas, creating π-chymotrypsin (active, but not fully functional). 3. π-chymotrypsin acts on other π-chymotrypsins- removing two dipeptides (2 peptide bonds per area for 2 different areas). π-chymotrypsin cleaves 2 peptide bonds b/w 13th & 16th aas and 2 peptide bonds b/w 146th & 149th aas. 4. This creates an active α-chymotrypsin w/ 3 diff. chains held together by disulfide bonds. These 3 chains are: A chain (1-13 aas), B chain (16-146 aas), C chain (149-245 aas). 5. Proteolysis of chymotrypsinogen causes local conformational change that allows the active site & oxyanion hole to form (needed to stabilize tetrahedral intermediate).

How is blood clotting regulated? (4)

1. Clotting must be precisely regulated 2. Specific inhibitors are critical for the regulation of clotting factors. 3. The most important inhibitor is antithrombin III, which inactivates thrombin by binding irreversibly to it. 4. Antithrombin III resembles α1-antitrypsin - these are members of the serpins (serine protease inhibitors).

What is elastase? How is it inhibited? What happens if there is too much elastase?

1. Elastase breaks down elastin to elastic fiber that interacts w/ collagen to determine mechanical properties of connective tissues. 2. α1-Antitrypsin is an inhibitor of elastase (not so much trypsin). It binds irreversibly to elastase and prevent its over-reacting. 3. Unrestrained elastase destroys alveolar walls in the lungs by digesting connective tissue proteins.

How is fibrinogen converted to fibrin clot? (6)

1. Fibrin monomer is made up of pairs of three kinds of chains: α2β2γ2. 2. Thrombin cleaves 4 bonds in the central region of fibrinogen- releasing an A peptide (18 residues) from each α and a B peptide (20 residues) from each β chain. 3. Releasing a total of 4 peptides [2 A peptides & 2 B peptides]. These A & B peptides are called fibrinopeptides. 4. A & B peptides are negatively charged, preventing interaction b/w fibrinogen monomers- so, they must be removed. 5. After thrombin cleaves these peptides, this creates a fibrin monomer. 6. Fibrin monomers then interact w/ each other spontaneously to form fibrin polymers, which are blood clots [mesh-like to seal off rupture in blood vessels].

Explain the basis of the blood clotting cascade.

1. Intrinsic pathway results from rupture of blood vessels 2. Extrinsic pathway results from tissue damage and releasing clotting substances in response to trauma (physical injuries) 3. Intrinsic & extrinsic pathways are triggered by diff. signals, but 2 pathways converge at the final common pathway. 3. Both pathways are precisely regulated & signals are amplified in both pathways. 4. Irreversible processes

What causes emphysema? (3)

1. Lack of α1-antitrypsin + subsequent alveolar walls destruction leads to emphysema. 2. People w/ emphysema have a hard time breathing. 3. Smoking increases likelihood of someone who is a heterozygote in the defect developing emphysema. Cigarette smoking oxidizes methionine 358 of the inhibitor, a residue critical for binding elastase. α1-antitrypsin can no longer bind to elastase --> leads tp excess elastase destroying alveolar walls.

What makes up blood clots? How are they held together?

Fibrin polymers; Covalent interaction (cross link of glutamine in one monomer and lysine in other monomers- catalyzed by transglutaminase, XIIIa) between α of one fibrin monomer and γ of another fibrin monomer.

Describe the extrinsic pathway of the blood clotting cascade.

Responds to physical injuries; quick. Thrombin activates factor 7 (VII), which joins w/ tissue factor (trauma) to activate factor 10 (X). This is where it converges w/ intrinsic pathway at the final common pathway.

Describe the intrinsic pathway of the blood clotting cascade.

Slightly slower than extrinsic; responds to rupture of blood vessels. Two enzyme can initiate this pathway: Kininogen & Kallikrein. These enzymes activate factor 12 (XII) --> activates factor 11 (XI) --> activates factor 9 (IX) --> joins w/ factor 8 (VIII) that is activated by thrombin to activate factor 10 (X). This is where it converges w/ extrinsic at the final common pathway.

Fibrin clots are lysed by plasmin that is activated by ________.

TPA