Exam 2 Study Questions Flashcards
How does warfarin prevent thrombosis?
It looks similar to the cofactor vitamin K and can replace it. It’s not reactive so the enzyme (thrombin) won’t work. It reduces thrombosis by reducing blood clots from forming inside of the body.
What causes hemophilia?
You are missing a disfactor that prevents Factor X from being activated
What is the Vitamin K dependent enzyme system?
It’s a system that’s dependent upon Vitamin K (the cofactor) that helps synthesize prothrombrin.
What does the Vitamin K dependent enzyme system do to prothrombrin?
The system converts the 1st 10 GLU residues in the N-terminal region of prothrombrin to (gamma) y-carboxyglutamate .
How does prothrombrin get to where it needs to be in the Vitamin K dependent system?
It binds to calcium at a high affinity. The calcium is on the phospholipid membrane of blood platelets and the they travel through the blood to the site of the wound.
How does prothrombrin associate with the calcium ions?
It associates to the (-) region on of the y-Carboxyclutamate residue. Ca2+ has a positive charge
How does protrhombrin disassociate with calcium ions?
Calcium is removed by being activated. This happens when Factor X cleaves prothrombrin into thrombin.
How is a soft clot formed?
Soft clots are formed from the and association of fibrin alpha domains interacting (cross linking) with fibrin gamma domains (globular domains).
How are soft clots stabilized?
Soft clots are stabilized with additional amide bonds between glutamine and lysine side-chains. The glutamine are on the beta subunit and the lysine are on the alpha subunit. Now there are covalent bonds formed from two different subunits that helps to stabilize the soft clot.
How can you prevent thrombosis?
By using clinical derivatives of coumarin that look similar to vitamin K. (dicoumarol & warfarin)
What does thrombin do to fibrinogen in terms of clotting?
Fibrinogen is converted by thrombin into a fibrin clot. Thrombrin cleaves off portions of the alpha & beta chains leaving them uncovered. When they are uncovered it allows them to associate better.
Define zymogen.
Zymogens are inactive enzyme precursors or proenzyems. The are apart of the digestive system. They are turned on in the small intestines and the enzymes breakdown polypeptides.
Where are zymogens made?
In the pancreas by acinar cells in response to a hormonal or nerve impulse.
Why is proteolytic cleavage important to achieve chymotrypsin enzyme activity?
Because cleaving leads to conformational change in the active site domain. Allows active site and an oxyanion hole to be formed. (Forms a cavity for substrate binding)
What is an isozyme and why are they important?
Isozyme or isoenzymes are enzymes that differ in amino acid sequence but catalyze the same reaction. There are different enzyme kinetics and are encoded by different genes. They allow you to selectively turn on an enzyme either in a given tissue or developmental time point. Allows for more control
Why is phosphorylation a powerful regulatory tool?
Phosphorylation adds two negative charges to the modified protein. The two negative charges can’t be close to each other and new electrostatic interactions form as old interactions break. This dramatically changes the conformation of the protein. The phosphoryl group can form 3 or more hydrogen bonds which also contributes to conformational change. It also brings free energy with it.
How do protein kinases vary in degree of specificity?
By using either a dedicated protein kinase or a multifunctional protein kinase
What is a dedicated protein kinase?
A dedicated protein kinase phosphorylates a single protein or closely related one. It phosphorylates a very specific substrate. Ex: Pyruvate
What is a multifunctional protein kinase?
It is a kinase that modifies many different types. It recognizes a consensus sequence of amino acids. It has a bunch of different targets as long as it has the right consensus sequence of amino acids.
What is a kinase & what does it do?
It is an enzyme that catalyzes the transfer of a phosphate group from ATP to a specified molecule. It phosphorolates things by taking the gamma phosphate (end) of ATP and transferring it to a protein. Always uses ATP and left with ADP.
What are types of kinases?
Serine/threonine and tyrosine
What is phosphatase?
A protein that reverses the effects of kinase. It removes a phosphate group and puts protein back into its original formation.
Why are the regulatory subunits so good at inhibiting the catalytic subunits of PKA?
Because it has a pseudosubstrate sequence that’s exactly the same as the substrate sequence but without the OH group. The substrate sequence contains a serine which gets phosphorylated and the pseudosubstrate contains an alanine thats has an nonreactive methyl group. The psedosubstrate forms the same interactions as the normal substrate with the active site. It’s only missing the functional part to be phosphorylated. The pseudo substrate hold the catalytic subunits inlace preventing them from phosphorylating.
How does PKA get activated?
A signal from outside of the cell signals turn on PKA. It does this by generating cAMP that binds to the regulatory subunits. Once it does that, they change shape (conformational change) by allowing the pseudosubunit domains to move out of the catalytic site allowing it to become active and bind to the substrate and phosphorylate.
How does PKA get turned off?
When cAMP levels drop in the cell, they let go of the regulatory subunits and they change back into their initial shape. Then they pair back up with the catalytic subunits.
Why is the pseudosubstrate so useful?
because it has the same reactions as regulatory substrate
What is the end point of an ATCase catalyzed reaction?
CTP production
What is feedback inhibition and how is it related to CTP?
A cellular control mechanism in which an enzyme that catalyzes the production of a particular substance in the cell is inhibited when that substance has accumulated to a certain level, thereby balancing the amount provided with the amount needed. The catalytic rate is fast at low concentrations of CTP but slows down with increased levels of CTP. (makes intermediate N-carbomoylaspartate)
How is ATCase inhibited and explain what it means?
It’s an allosteric enzyme and it’s allosterically inhibited. An allosteric enzyme displays sigmoidal kinetics. Aspartate is an allotter effector and it binds to the catalytic trimer. There are multiple subunits and more than one binding spot.
What is the makeup of ATCase?
ATCase consist of separable catalytic and regulatory subunits. There are 3 regulatory dimers (6 total) that interact with 2 catalytic trimers. In the regulatory subunit there is a zinc that is required to hold them in place and prevent them from disassociating.
Describe the active site of ATCase.
It lies at the interface of two catalytic subunits. It is made up of amino acids from the subunits itself as well as amino acids of the neighboring subunit.
How does the the active site influence the T & R state of ATCase?
When the substrate binds to the active site, ATCase shifts from a T to R state. In the R state there is a high affinity for substrate and catalysis. In the absence of substrate there is a low affinity for substrate and catalysis. In the relaxed state, bonds are holding the enzyme back and it’s able to catalyzes.
How does CTP inhibit ATCase?
CTP is an allosteric regulator of ATCase. It’s a noncompetitive inhibitor. It inhibits ATCase by binding to the regulatory subunits and keeping the enzyme in the T state (tense) which is the low activity state. It keeps the catalytic subunits in a T state. It doesn’t compete with the substrate. It binds to another site that effects how the active sites work.
How does ATP affect ATCase?
ATP competes with CTP for binding to regulatory sites. At high levels of ATP, it prevents CTP from binding. ATP puts the enzyme into a R state. ATP indicates there is a high level of GTP (increased levels of purines which means it needs to makes pyrimidines). It’s an allosteric activator. It’s a noncompetitive regulator. It’s telling the enzyme you need to make more CTP.
Name the 3 amino acids that make up a catalytic triad for a serine protease.
Aspartate, histidine & serine
What is a tetrahedra intermediate?
Tetrahedral intermediates result from nucleophilic addition to a carbonyl group.
What’s an oxyanion hole?
It stabilizes the transition state. It consists of glycines that goes up over the active site and allowing hydrogen bonds to form between O- on the tetrahedral intermediate and stabilizing it on the chymotrypsin reaction. (Interactions with -NH groups)
What is the S1 pocket?
A deep hydrophobic pocket that can accommodate long, uncharged side chains. Binding of substrate into the pocket positions the adjacent peptide bond into the active site for cleavage. It determines specificity.
How does the S1 pocket of serine protease dictate the type of substrate used?
There are there different pocket types and each one dictates what can fit inside of them.
Chymotrypsin: Is a long deep hydrophobic pocket that can accommodate larger hydrophobic amino acids like tryptophan, phenlyalanine, tyrosine, or methionine.
Trypsin: Is a deep pocket with an aspertate ad the bottom giving it a negative charge. It will accommodate long positively charged functional groups like lysine and arginine.
Elastase: Has valines sticking out the side of it’s pocket making it more shallow. It can accommodate smaller hydrophobic functional groups like valine, alanine or leucine.
What is required for for catalytic activity of carbonic anhydrase? What is in it’s active site?
Zinc - it’s a cofactor (metal ion catalysis) Histitdines
How does water get deprotonated in the catalysis of carbonic anhydrase?
When water binds to zinc, it lowers the pKa from 15.7 to 7. (Normal physiological pKa is 7.4). At this low pKa, water loses a proton at neutral pH to generate a hydroxide anion.
Describe the reaction of carbonic anhydrase & why is it important?
The OH group is going to attack the CO2 and then essentially water comes in and displaces bicarbonate (HCO3). It’s coordinated and interacting with the zinc. Water comes in and displaces bicarbonate and the enzyme is regenerated for another round. A proton shuttle facilitates rapid regeneration of the active form of the enzyme. The histidine shuffles the proton to the buffer so that it can’t reprotonate the water. Important because without this you may not have catalysis. If the proton was left there, it could reprotonate and go back to water not allowing the -OH group on the active sit of carbonic anhydrase to work and prevent catalysis. It can’t do it’s job if it doesn’t have a reactive protein.
What doe restrictions enzymes do?
Restrictions enzymes perform highly specific DNA-cleavage reactions. They cleave DNA backbone (within the strand) at specific recognition sites (sequences)
What kinds of bonds to restrictions endonucleases break?
phosphodiester bonds (cuts between two nucleotides)
What is held together within the enzyme that hydrolyzes the phosphodiester bond?
Water is held by Mg2+ ( its the cofactor)
What is a scissile bond?
It’s the bond to be cut
How does DNA become distorted within the active sites of restriction endonucleases?
Once you have the correct hydrogen bonds forming it kinks the DNA into the active sites.
How does methylation affect restriction enzymes?
Methylation prevents the host DNA from interacting with the enzyme and preventing the enzyme from cutting its own DNA.
How do enzymes affect activation energy?
They lower it because they lower the energy required to get to the transition state.
What is an ES complex?
Substarte-Enzyme Complex Specificity of substrate binding depends on the precisely defined arrangement of the atoms in the active site
