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What does the appearance of specific enzymes & proteins indicate?
organ damage, because enzymes are expressed only in certain tissues but not others
eg. Liver Damage: a liver enzyme that balances amino acids levels is alanine transaminase (ALT); its appearance in the blood is indicative of cell damage
Alanine Transaminase (ALT)
- presence of this liver enzyme in the blood stream is indicative of liver damage
- (catalyses the interconversion of alanine & pyruvate)
What is another indicator of liver damage?
low alpha-1 antitrypsin
- • is normally secreted by liver & taken up by the lung
- • if absent from the blood, liver isn’t functioning properly (because it normally should be there)
an inactive enzyme precursor
- • concept: an enzyme's active site is blocked or its residues are out of alignment until proteolytic cleavage occurs at a specific activation site on the chain
- • when triggered proteolytic cleavage occurs rapidly → resulting in nearly instant enzyme activation
The Blood Clotting Cascade
- • 1st triggered by a break in the lining of a blood vessel wall
- • this exposes negatively charged lipid molecules
- • only a few molecules will recognize the damage
- • the final aspect of coagulation is formation of a fibrin clot
- • point is there are many steps: this is a cascade where an increasing number of proteins are involved
- • prothrombin is cleaved on a membrane surface by another enzyme to make thrombin
How much fibrinogen is there in 1 cubic centimeter of blood?
- • once thrombin cleaves peptides off fibrinogen, it becomes Fibrin
- • Fibrin spontaneously forms monomers
- - this creates an initial loose clot
- • then cross-linking occurs to result in a stable clot
- • the fibrin clot forms a mesh like substance that holds together RBCs & aids in the blood clotting process
What previously discussed enzyme is related to Thrombin?
Chymotrypsin; thrombin has many of the same features because it’s also a serine protease
- abundant conversion enzyme in the blood coagulation cascade
- • thrombin is 1st made as a larger precursor
- • it’s important for it to be sitting on membrane surface for it to be activated
- • upon cleavage (2 peptide bonds are broken) thrombin is released
- • substrate of thrombin is fibrinogen, which is a precursor of fibrin
- • fibrin is what forms the blood clot
performs 2 proteolytic cleavages that turn prothrombin into its active form, thrombin
components of the prothrombin-converting complex: (4)
- 1) calcium
- 2) phospholipids
- 3) Factor V (cleaves prothrombin to prethrombin)
- 4) Factor X (cleaves prethrombin to thrombin)
- (3 & 4 are blood coagulation proteins)
formed after a single proteolytic cleavage by prothrombin-converting complex; a 2nd cleavage between residues 15 & 16 by the same complex results in thrombin
What happens to Prethrombin after cleavage between residues 15 & 16?
- • light chain residues = 1-15 don't change much; they're held to the rest of the enzyme (heavy chain) by a disulfide bond
- • in the active enzyme, residue isoleucine 16 reaches over & forms a stabilizing interaction with residue aspartic acid 194
- • this repositioning results in the proper formation of the catalytic triad’s serene protease nucleophile residue, serine 195
What is the relationship between the amino acid isoleucine 16 & aspartic acid 194 in the serine protease enzyme Thrombin?
- • aspartic acid 194 is adjacent to the catalytic residue serine 195
- • aspartic acid 194 (as well as serine 195 subsequently) is held in place by a hydrogen bond between it & isoleucine 16
- • if the hydrogen bond is broken, the peptide undergoes a conformational change causing serine 195 to no longer be active
- • without that hydrogen bond the enzyme will be inactive
Where in the cell is the mature form of Thrombin synthesized?
- • on the cell membrane surface but it's water soluble
- • thrombin activation is calcium DEPENDENT
- • the γ-carboxy-glutamate binds metal ions, including calcium
- • the whole process of enzyme activation can be controlled by plasma membrane binding
Prothrombin is modified by γ-carboxylation of which amino acid residue on its N-terminus?
Why is it important for prothrombin to be γ-carboxylated?
it allows for calcium binding, which is required to anchor prothrombin to the cell membrane
(calcium ions binding to prothrombin carboxyglutamate mediate the process of attaching prothrombin to blood vessel endothelium)
Does γ-carboxylation of thrombin regulate enzyme activity?
γ-carboxylation is a post-translational modification that DOES NOT directly regulate enzyme activity, but IS required for formation of the active enzyme for blood coagulation
What does Prothrombin need in order to bind calcium allowing it to form a structure that can bind a membrane surface?
an amino acid modification: glutamate → γ-carboxyglutamate by Carboxylase/Vitamin K complex
a cofactor required for the gamma-carboxylation of prothrombin
What would vitamin K antagonists (eg. Dicoumarol) do?
- interfere with the carboxylation reaction
- • poorly carboxylated Prothrombin & it's activating enzyme do not bind well to the membrane surface → reducing activation
- • Dicoumarol behaves like Vitamin K but doesn’t allow it to act as a cofactor & participate in the reaction
• a vitamin K analogue (synthetic derivative of dicoumarol) that is used to treat excessive blood clotting (thrombosis)
• Vitamin K ANALOGUES interfere with the carboxylation reaction
How does Warfarin work?
• it inhibits the γ-carboxylation reaction of Prothrombin, thereby reducing the number of Prothrombin molecules attached to the cell membrane
• this reduces the amount of active thrombin that can be synthesized by a cell
- a protein floating around in the blood that occupies (tightly complexes with) some amount of thrombin & inhibits it from causing coagulation
- • these inhibitors bind so tightly that the enzyme-inhibitor complex is degraded as a whole
- • inhibition is irreversible
- an anti-coagulant that promotes the high-affinity binding of antithrombin to thrombin → reducing blood clotting (reduces the amount of thrombin available for blood coagulation)
- • fast acting, while warfarin is slow to cause an effect
- • a type of serine protease released by neutrophils whose purpose is to neutralize foreign particles
- • when released in the lung it also breaks down elastin fibers
- • unregulated elastase activity would result in emphysema
What normally exists in tissue to prevent over-zealous elastin breakdown by elastase?
- α1-antitrypsin (or α1-antiprotease): an elastase inhibitor
- • a mutation in α1-antitrypsin means the inhibiting reaction doesn’t occur efficiently
- • this results in uninhibited elastase, which will chew on the elastin of the lung → scarring lung tissue → problems breathing
α1-antitrypsin in the lungs
- • an irreversible elastase inhibitor that prevents neutrophil-produced elastase from degrading elastin fibers
- • individuals with mutated α1-antitrypsin often have lung destruction & emphysema
How does cigarette smoking affect the activity of α1-antitrypsin?
- • cigarette smoke oxidizes a methionine residue on α1-antitrypsin involved in binding elastase
- • methionine → methionine sulfoxide
- • oxidation of this residue PREVENTS inhibition of elastase
- • this results in over-degradation of lung elastin fibers → lung scarring & emphysema
How can oxidation of α1-antitrypsin caused by smoking be treated?
- intravenous administration of α1-antitrypsin
- • the protein diffuses from blood into lung, where it reaches therapeutic levels in the fluid surrounding epithelial cells
- • (people with a deficiency of α1-antitrypsin shouldn't smoke because they will be extremely sensitive to the action of elastase)
What can protein phosphorylation lead to?
- phosphorylation or dephosphorylation changes the charge of an amino acid residue
- • eg. serine, threonine, or tyrosine have -OH residues; adding a phosphate group to them will cause them to adopt a negative charge
- • if one of those amino acids is in or near the active site of an enzyme, the enzyme activity may be changed
- *changing the charge can change the enzyme activity*
Kinase v. Phosphatase
- kinase: puts ON the phosphate
- phosphatase: removes the phosphate
What does phosphorylation in the activation loop of a kinase lead to?
1000 fold increase in catalytic activity
remainder of questions come from previous BC flashcards
phosphorylation or dephosphorylation modifies the ______ of an amino acid residue
charge; if said amino acid residue (serine, threonine, or tyrosine) is in or near the active site of an enzyme, the enzyme activity MAY be changed
Which amino acids can typically be modified by enzymatic phosphorylation/dephosphorylation?
serine, threonine and tyrosine
How does phosphorylation of a kinase alter it's catalytic abilities?
phosphorylation, specifically in the activation loop of a kinase, leads to a 1000 fold increase in catalytic activity (eg. increases substrate binding)
- reversibly compete with a substrate for an enzyme's active site
- usually resemble the substrate in structure
- they are able to fit into the active site but cannot be acted upon by the enzyme
- eg. ethanol is a competitive inhibitor of ethylene glycol
- (binding site only)
- competitive inhibition; Vmax reached eventually but it takes much more substrate than usual
with competitive inhibitors:
the more you use, the more COMPETITION there is with substrate
- Ki measures the affinity of an enzyme for a certain inhibitor
- the SMALLER the Ki, the GREATER the inhibitor's effectiveness
What is the effect of competitive inhibitors on Km and Vmax?
Competitive inhibitors increase Km but DO NOT alter Vmax
Km measures the affinity of an enzyme for its substrate. The smaller the Km, the greater the enzyme's affinity for its substrate. Km is also defined as 1/2 Vmax.
- bind to the enzyme in a location OTHER THAN the active site (whether or not a substrate is present) & affect the amino acid residues of the enzyme involved in the catalysis of the reaction
- (catalytic machinery only)
- noncompetitive inhibition; Vmax never achieved
What is the effect of noncompetitive inhibitors on Km and Vmax?
Noncompetitive inhibitors LOWER Vmax but do NOT alter Km
using noncompetitive inhibitors, Vmax:
CANNOT be reached, no matter how much substrate is added
- form very tight complexes, kill the enzyme by forming covalent bonds with it
- eg. DIFP
an irreversibly inhibitor that inhibits serene proteases, specifically the enzymatic hydrolysis of acetylcholine (NT) by acetylcholine esterase, thus acting as an effective nerve gas
recovery from reversible inhibitors:
related to half-life (hours)
recovery from irreversible inhibitors:
the effective time relevant for recovery from irreversible inhibitors is the time required for re-synthesis of an enzyme
What are enzyme activators?
positive effectors that promote an enzyme to exist in its active form where it is a more effective catalyst
True or False: allosteric enzymes often catalyze the first step of a reaction sequence?
What does an allosteric enzyme curve look like?
What is meant by the term 'feedback inhibition'?
Feedback inhibition is a phenomenon by which the end product of a reaction or sequence of reactions inhibits the enzyme or enzymatic cascade. When sufficient product is produced, the system automatically turns itself off.
Phosphofructosekinase 1 (PFK1)
- enzyme responsible for phosphorylating fructose-6-phosphate (using ATP) in the committed step of glycolysis
- -is allosterically activated by AMP (signals that cell is energy poor)
- -is reversibly inhibited by ATP (product of glycolysis)
List three common characteristics of committed steps:
- 1. They are functionally irreversible
- 2. They occur early in the enzymatic pathway
- 3. They occur in only one pathway
the activation of thrombin is:
Which enzyme catalyzes the conversion of soluble fibrinogen to insoluble fibrin?