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· Many enzymes acquire full __ as they spontaneously fold into their characteristic 3D forms. Some, however, are still inactive until __= __ or __
o __is not needed for cleavage, allowing even __ to be activated
o This cleavage takes place how many times?
- enzymatic activity
- the cleavage of one or a few specific peptide bonds
- zymogens or proenzymes
- ATP
- cells outside
- just once in the life of an enzyme molecule
-
· Some examples:
- digestive enzymes
- blood clotting
- hormones
- o Digestive enzymes that hydrolyze proteins are zymogens in the stomach and pancreas
- o Blood clotting is mediated by a cascade of proteolytic activations that ensures a rapid and amplified response to trauma
- o Some protein hormones are made as inactive precursors, like insulin (proinsulin)
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· Some examples:
- collagen
o Many developmental processes are controlled by the __
o Programmed cell death, or __, is mediated by __, which are synthesized in precursor form as __.
- Collagen is derived from procollagen
- activation of zymogens
- apoptosis
- proteolytic enzymes called caspases
- procaspases
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· Chymotrypsin is what?; and, when inactive, it is __
o The __ and __ are made in the __ of the pancreas and stored inside __. The __ accumulate at the __; when the cell is stimulated by a __ or a __, the contents of the __are released into a __ leading to the __
- a digestive enzyme that hydrolyzes proteins in the small intestine
- chymotrypsinogen
- enzymes and zymogens
- acinar cells
- membrane bound granules
- zymogen granules
- apex of the acinar cell
- hormonal signal or a nerve impulse
- granules
- duct leading into the duodenum
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· __, a single polypeptide chain consisting of 245 amino acid residues, is devoid of __. It is converted into a fully active enzyme when the __.
The resulting active enzyme, called __, then acts on other __ molecules by removing __ to yield __, the stable form of the enzyme. The three resulting chains in __ remain linked to one another by two __. The striking feature of this activation process is that what happens?
- Chymotrypsinogen
- enzymatic activity
- peptide bond joining arginine 15 and isoleucine 16 is cleaved by trypsin
- pi-chymotrypsin
- pi-chymotrypsin
- two dipeptides
- alpha-chymotrypsin
- alpha-chymotrypsin
- interchain disulfide bonds
- cleavage of a single specific peptide bond transforms the protein from a catalytically inactive form into one that is fully active
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· Proteolytic activation of chymotrypsinogen leads to the formation of a __
o The cleavage triggers key __
§ The newly formed __ turns inward and forms an __ in the interior of the __molecule
- substrate-binding site
- conformational changes
- amino-terminal group of isoleucine 16
- ionic bond with aspartate 194
- chymotrypsin
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This __ triggers a number of conformational changes. __ moves from a deeply buried position in the __to the surface of the __, and residues 187 and 193 move farther apart from each other. These changes result in the formation of the __. One side of this site is made up of __. This cavity for binding part of the substrate is not __
- electrostatic interaction
- Methionine 192
- zymogen
- active enzyme
- substrate-specificity site for aromatic and bulky nonpolar groups
- residues 189 through 192
- fully formed in the zymogen
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§ The __ in catalysis by chymotrypsin is stabilized by __ and __. One of these NH groups is not appropriately located in chymotrypsinogen, and so the __ is incomplete in the __
- tetrahedral transition state
- hydrogen bonds between the negatively charged carbonyl oxygen atom of the substrate and two NH groups of the main chain of the enzyme
- oxyanion hole
- zymogen
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§ The conformational changes elsewhere in the molecule are very small. Thus, the __ of __ in a protein can be accomplished by discrete, highly localized conformational changes that are triggered by the __
- switching on
- enzymatic activity
- hydrolysis of a single peptide bond
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· The generation of __ from __ leads to the activation of other __
o The structural changes accompanying the activation of __are different from __
o Four stretches of polypeptide, constituting 15% of the molecule, changes markedly on __
§ These regions are flexible in the __, whereas they have a well-defined conformation in __.
§ The oxyanion hole in trypsinogen is too far from histidine 57 to promote the formation of the tetrahedral transition state
- trypsin from trypsinogen
- zymogens
- trypsinogen
- chymotrypsinogen
- activation
- zymogen
- trypsin
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· The digestion of proteins in the __requires the concurrent action of several __, because each is specific for a limited number of side chains.
o Thus, the __must be switched on at the same time
o __ is achieved by the action of trypsin as the common activator of all the __
- duodenum
- proteolytic enzymes
- zymogens
- Coordinated control
- pancreatic zymogens
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· To produce active trypsin, the cells that line the duodenum secrete an enzyme, __, which hydrolyzes a unique __ in __ as the __enters the __from the __. The small amount of trypsin produced in this way activates more __ and other __.
o Thus, the formation of __by __is the __.
- enteropeptidase
- lysine-isoleucine peptide bond in trypsinogen
- zymogen
- duodenum
- pancreas
- trypsinogen and the other zymogens
- trypsin
- enteropeptidase
- master activation step
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· Some proteolytic enzymes have __
o The conversion of a __into a protease by cleavage of a single peptide bond is a __; but, its __
o So, to stop __, __ are used
- specific inhibitors
- zymogen
- precise means of switching on enzymatic activity
- irreversible
- proteolysis
- protease inhibitors
-
§ Ex: __ inhibits __by doing what?; it is a very effective __ that lies in the __ of the enzyme, positioned such that the __ interacts with the __.
- pancreatic trypsin inhibitor
- trypsin
- binding very tightly to its active site
- substrate analog
- active site
- side chain of lysine 15 of the inhibitor
- aspartate side chain in the specificity pocket of trypsin
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· In addition, there are many __ between the __ and that of its __·
The __ and surrounding atoms of the inhibitor fit snugly in the active site of the enzyme
- hydrogen bonds
- main chain of trypsin
- inhibitor
carbonyl group of lysine
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§ The structure is essentially __on binding to the enzyme. Thus, the inhibitor is __into a structure that is __
§ The inhibitor is a __, but its structure complements the __, allowing __
- unchanged
- preorganized
- highly complementary to the enzyme’s active site
- substrate
- enzyme’s active site
- tight binding
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· A trypsin inhibitor exists because __
· __ also protects tissues from digestion by __; it blocks __more effectively than it blocks trypsin
- trypsin activates other zymogens. So, to prevent a premature cascade from occurring, the trypsin inhibitor needs to bind to prevent severe damage to tissues
- Alpha1-antitrypsin
- elastase
- elastase
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What does alpha1-antitrypsin do?
o It blocks the action of target enzymes by binding nearly irreversibly to their active sites. Deficiencies or errors in this can cause emphysema
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· Enzymatic cascades are often employed in biochemical systems to achieve a rapid response.
o In a cascade, what happens?
o Two means of initiating blood clotting:__
They both ___
an initial signal institutes a series of steps, each of which is catalyzed by an enzyme, amplifying the signal at each step
- intrinsic pathway
- extrinsic pathway
form a clot composed of fibrin
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Intrinsic pathway
active by exposure of anionic surfaces on rupture of the endothelial lining of the blood vessels
-
Extrinsic pathway
§ initiated when trauma exposes tissue factor, an integral membrane glycoprotein, generating small amounts of thrombin, which amplifies the clotting process by activating enzymes and factors that lead to the generation of yet more thrombin
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· __ converts __ to __. __is made of __ connected by __ and has __, 2 each of __.
o the rod regions are __, a recurring motif in proteins
o Thrombin cleaves for __ peptide bonds in the __. On cleavage, an __ is released from each of the two Aa chains as is a B peptide of 20 residues from each of the two Bb chains
- Thrombin converts fibrinogen to fibrin
- Fibrinogen
- three globular units
- two rods
- six chains
- Aa, Bb, and y
- triple stranded alpha-helical coiled coils
- arginine- glycine
- central globular region of fibrinogen
- A peptide of 18 residues
-
§ these A and B peptides are __; and, if lacking one, it is a __
· __ spontaneously do what?
o the homologous__ and __ have __at the __ ends, which have __ that interact with the peptides
- fibrinopeptides
- fibrin monomer
- fibrin monomers
- assemble into ordered fibrous arrays called fibrin
- B and gamma chains
- globular domains
- carboxyl-terminal
- binding holes
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§ B domain: specific for __, where the y domain binds __.
· Exactly these sequences (__) are exposed at the __ of the __ and __ chains, respectively, on __.
o The __of the alpha subunits fit into the __on the gamma subunits of another monomer to form a __, which extends when the __of the B subunits fit into the __of B subunits of other protofibrils
- H3N+-Gly-His-Arg
- H3N+-Gly-Pro-Arg-
- knobs
- amino-terminal ends
- B and alpha chains
- thrombin cleavage
- knobs
- holes
- protofibril
- knobs
- holes
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o Therefore, peptide-bond cleavage exposes what?. The newly formed “__” is stabilized by the __ between the __
§ This cross linking reaction is catalyzed by __, which is activated from the __form by __
- new amino termini that can participate in specific interactions
- soft clot
- formation of amide bonds
- side chains of lysine and glutamine residues in different monomers
- transglutaminase
- protransglutaminase
- thrombin
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· __is readied for activation by a __
o Inactive prothrombin contains __major domains, with the __ at the __
§ First domain: __
§ Second and third: __
§ These domains work in concert to keep __in an __form and to target it to appropriate sites for its activation by __ and __
- Prothrombin
- vitamin K-dependent modification
- four
- serine protease domain at the carboxyl terminus
- gla domain
- kringle domains
- prothrombin
- inactive
- factor Xa ( a serine protease)
- factor Va (a stimulatory protein)
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o Activation is begun by __to do what?; cleavage of the bond between __ and __ yields __
- proteolytic cleavage of the bond between arginine and threonine
- release a fragment containing the first three domains
- arginine and isoleucine
- active thrombin
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· A lack of Vitamin K results in __
o Studies show the importance of this
§ __is an anticoagulant used to prevent thrombosis in patients prone to __.
· Normal prothrombin contains __
- defective blood coagulation
- Dicoumarol
- clot formation
- gama-carboxyglutamate
-
o The vitamin K-dependent __converts __, a weak __, into __, a much stronger chelator
§ __is thus able to bind __, which does what?
- carboxylation reaction
- glutamate
- chelator of Ca2+
- gamma-carboxyglutamate
- Prothrombin
- Ca2+
- anchors the zymogen to phospholipid membranes derived from blood platelets after injury
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§ The binding of __to __ is crucial because it does what?
· The __ is removed during __, doing what so that it can what?
- prothrombin
- phospholipid surfaces
- brings prothrombin into close proximity to two clotting proteins that catalyze its conversion into thrombin
- calcium binding domain
- activation
- freeing the thrombin from the membrane so that it can cleave fibrinogen and other targets
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· __ is a sex-linked recessive characteristic, in which __ of the intrinsic pathway is missing or has reduced activity
o __ stimulates activation of __, the final protease of the intrinsic pathway, by __
Activation is impaired
- Hemophilia A
- factor VIII
- Factor VIII
- factor X
- factor IXa, a serine protease
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· The clotting process must be precisely regulated
o Clots must do what?
o Activated factors are __ because they are __
o Thrombin has a dual function:__
form rapidly yet remain confined to the area of injury
short lived
diluted by blood flow, removed by the liver, and degraded by proteases
it catalyzes the formation of fibrin and it initiates the deactivation of the clotting cascade
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· Specific inhibitors of clotting factors are also critical
o __: inhibits complex of TF-VIIa-Xa
o __: a plasma protein that __ by __; resembles __ except that it __ much more strongly than it __; it also blocks other serine proteases in the clotting cascade
- Tissue factor pathway inhibitor
- Antithrombin III
- inactivates thrombin
- forming an irreversible complex with it
- aa-antitrypsin
- inhibits thrombin
- inhibits elastase
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§ The inhibitory action of __ is enhanced by __, a negatively charged polysaccharide found in __ near the walls of BVs and on the surfaces of endothelial cells
o __acts as an __by doing what?
§ __ and __ are __, a family of serine protease inhibitors
- antithrombin III
- heparin
- mast cells
- Heparin
- anticoagulant
- increasing the rate of formation of irreversible complexes between antithrombin III and the serine protease clotting factors
- Antitrypsin and antithrombin
- serpins
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· Clots are not __ but are designed to __when the structural integrity of damaged areas is restored.
o Fibrin is split by __, a serine protease that __ and can __
- permanent structures
- dissolve
- plasmin
- hydrolyzes peptide bonds in the coiled-coil regions
- diffuse through aqueous channels in the porous fibrin clot to cut the accessible connector rods
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§ Plasmin is formed by the __, an inactive precursor that has a high affinity for the fibrin clots.
· This conversion is carried out by __
- proteolytic activation of plasminogen
- tissue-type plasminogen activator (TPA)
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o However, a domain that targets __ to fibrin clots replaces the __. The __bound to fibrin clots swiftly activates __.
o In contrast, __activates __ very slowly.
o The gene for __has been cloned and expressed in cultured mammalian cells
- TPA
- membrane-targeting gla domain of prothrombin
- TPA
- adhering plasminogen
- TPA
- free plasminogen
- TPA
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