Enzymes 2 Flashcards
Isozyme
- Enzymes that catalyse the same reaction
- Multiple forms in an organism-sometimes in different tissues
- Products of different genesHow can isoenzymes be styd
What reaction do glucokinase and hexokinase catalyse?
Glucose + ATP > Glucose-Phosphate + ADP
What happens when blood glucose increases?
-Glucokinase activity increases but hexokinase activity does not respond as it is already working at Vmax.
What happens when blood glucose decreases?
-Gluconeogenesis releases glucose from the liver, but glucokinase cannot catalyse glucose back into glucose-6-phosphate under these conditions allowing glucose to be used in the body.
How can isoenzymes be studied?
Using electrophoresis
What is creatine kinase?
A dimer made from 2 polypeptides B and M
What does elevation of CK2 indicate?
Myocardial infarction
How are enzymes used in diagnosis?
- Measure activity and compare with normal
- Separate different forms of enzymes by electrophoresis and examine pattern
What does catalysing a reaction with 2 or more substrates usually involve?
Transfer of groups from one substrate to the other
How can transferring of groups between substrates occur?
- Random order or Ordered with ternary complex
- No ternary complex formation
What does lactate dehydrogenase exhibit to its catalysis of pyruvate to lactate?
An ordered sequential mechanism
Describe the catalysis of pyruvate by lactate dehydrogenase.
-Coenzyme (NADH) binds first and the lactate is always released first
What happens in an ordered sequential reaction mechanism?
The enzymes exists in a ternary complex, first with the substrates of the reaction and then (after catalysis) with the products of the reaction
What does creatine kinase exhibit when it catalyses the formation of phosphocreatine from creatine?
Random sequential mechanism
What happens in a random sequential mechanism?
The order of binding and release of substrate and product is random but the formation of a ternary complex still occurs first with substrates and then the products of the reaction
What are classic examples of reactions which have no ternary complex formation??
Reactions where amino groups are shuttled between amino acids and ketoacids
Where does the term ‘ping pong’ come from?
Substrates bounce on and off the enzyme as they are catalysed
What do allosteric enzyme not follow?
M-M Kinetics
Describe the kinetics of allosteric enzymes.
- One substrate binding to an enzyme subunit can cause changes in other active sites on other subunits.
- This can lead to cooperative binding of substrate molecules
What are allosteric enzymes composed of?
Many subunits which contain man active sites
What affects the activity of an enzyme?
- Temperature
- pH
- Inhibitors
How does temperature affect enzyme activity?
Increase:
- Increases molecule collisions
- Increases energy of molecules
- Will eventually denature enzyme
How does pH affect enzyme activity?
- pH changes the charge of amino acids
- If the active site amino acids charge changes the enzyme will cease to function
- Extreme pH will denature most enzymes
- pH will also affect the substances of the reaction, some of which may require H or OH groups to be involved in the reaction
What are the 3 kinds of enzyme inhibitor?
- Competitive inhibitor
- Non-competitive inhibitor
- Uncompetitive inhibitor
Competitive inhibitors
Bind to enzymes non-covalently and will usually resemble the substrate molecule, therefore competing with the active site
What do competitive inhibitors lead to?
-Decrease in affinity between active site and substrate, so the Km of ES increases
What can overcome competitive inhibition?
Increasing substrate concentration
What is Azidothylmidine?
A controversial drug created in the 90s for AIDs
How does AZT act?
- Acts by competitive inhibition of the reverse transcriptase enzyme
- Reverse transcriptase is used by HIV to produce dsDNA molecule from its ssRNA
- AZT undergoes triphosphorylation in the body and thus mimics the ordinary DNA precursor thymidine triphosphate
What is a transition state analogue?
An inhibitor that mimics the transition state
What is an example of a transition state analogue?
Oseltamivir (Tamiflu)
How does oseltamivir work?
- In the body oseltamivir is hydrolysed in the liver to its active form which is then able to block the activity of neuraminidase enzyme.
- Neuramindase normally cleaves sialic acid that allows the release of new virus particles form the cells
What is the concept of catalytic antibodies?
- Antibodies can be generated against potentially any biological molecule, therefore and antibody could be made that was specific to a transition state molecule.
- This would allow the production of an antibody with a structure that resembles that of the active site of the original enzymes
What difficulty is there with catalytic antibodies?
Transition states are notoriously difficult to isolate as they are the intermediate step between and ES an EP
In what disease would you find a naturally occurring catalytic antibody?
Lupus erythematosus
-Characterised by the autoantibodies attacking the connective tissue of the joints, skin, kidneys, heart and lungs
What are non-competitive inhibitors?
Inhibitors that bind to enzymes non-covalently and will usually attach to a site other than the active site of the enzyme
Why does the Km of the ES remain unchanged with non-competitive inhibitors?
The substrate is usually still able to bind to the active site
Why does Vmax decrease with non-competitive inhibitors?
Increasing substrate concentration does not change the inhibition.
What is an irreversible inhibitor?
An inhibitor that will usually bind to an enzyme covalently
What is a regulatory enzyme?
Key enzyme in a pathway
What are the 2 main ways regulatory enzymes modulate reactions?
- Allosteric enzymes
- Covalently modified enzymes
What is feedback inhibition?
A build up of end product of a pathway, or key junction in a pathway can ultimately slow the entire pathway
What can inhibit bacterial threonine dehydratase?
The final product of a 5 step pathway (L-isoleucine)
How does L-isoleucine inhibit bacterial threonine dehydratase?
It binds to a site other than the active site which changes the conformation of the active site blocking the enzymes action
How do allosteric enzymes work?
They bind non-covalently to a site other than the active site.
- This changes the enzymes structure
- Some effectors are activators
- Binding of the effector will increase or decrease the efficiency of substrate binding and processing
What are allosteric enzymes examples of?
Non-competitive inhibitors
What does the kinetics of allosteric enzymes suggest they do?
Increase of [S] a low levels has less of a response than the same increase at a higher level.
-This suggest that at low [S] it sensitises the enzyme
What are the 2 models?
- Concerted model
- Sequential model
Describe the concerted model.
- Each sub-unit can exist in 2 different conformations
- One binds substrate well the other doesn’t
- With no substrate the enzyme flips between the 2 conformations
- All sub-units must be in the same conformation
What do allosteric enzymes do in concerted model?
- They will stabilise the open conformation allowing S to bind more effectively
- They will stabilise the closed conformation and make it difficult for S to bind effectively
What does the sequential model assume?
- No flipping between different conformation states
- Sub-units exist in a conformation that can bind S, activators, inhibitors
- It is the binding that causes a conformational change
What does substrate binding in sequential model do?
Causes a change in one sub-unit
How can enzymes be regulated?
Through reversible covalent modification
What is a common covalent medication?
Phosphorylation
Where can proteins be phosphorylated?
- At a single site
- Multiple sites
- Multiples phosphorylations at one site
What enzymes are involved in phosphorylation?
- Protein kinases: add phosphate groups to proteins
- Protein phosphatases: remove phosphate groups from proteins
What do multiple phosphorylation sites allow?
Very fine control of enzyme function depending on the requirement of the particular enzyme at a given time
Other than in its active state, what other state can an enzyme exist in?
-Inactive precursor called a proenzyme
How do proenzymes become active?
They can be cleaved by protesases
What do digestive enzymes tend to be called?
Zymogens
