Enzymes 2 Flashcards
What do enzyme inhibitors do? What do they include?
- Enzyme inhibitors decrease the rate of the reaction
- Maybe reversible or irreversible
Enzyme inhibitors may be
vDrugs
vToxins
What is the most current drug therapy based on? Give example! What are the two types of enzyme inhibitors?
•Most of the current drug therapy is based on inhibition of enzymes. Eg: Penicillin/amoxycillin –inhibits the enzyme transpeptidase involved in bacterial cell wall synthesis
•Types:
–Irreversible inhibition -> inhibitor forms covalent bonds with enzymes Eg: Nerve agent Sarin for Acetylcholinesterase
**–Reversible inhibition -> noncovalent bonds
•Competitive inhibition
•Noncompetitive inhibition
•Uncompetitive inhibition **
What are the subcategories under reversible inhibition?
- Competitive inhibition
- Noncompetitive inhibition
- Uncompetitive inhibition
Explain the competitive inhibitor! How does the MM plot and the LB curve look like?
A competitive inhibitor
- Resembles the structure of the substrate (substrate analog)
- Binds to the active site on the free enzyme (competes with the substrate for active site)
- Increases the Km of the enzyme for the substrate
- Inhibition can be reversed by increasing substrate concentration
- Vmax remains unchanged (at sufficiently high [S], original Vmax can be achieved)
Malate competitively inhibits succinate dehydrogenase
The following are the enzymes, give thhe substrate, inhibitor and the significance
Dihydrofolate reductase
Angiotensin converting enzyme (ACE)
Dihydropteroate synthase
Vitamin K epoxide reductase
HMG CoA reductase
What happens in Noncompetitive inhibition ? What are some examples of noncompetitive inhibitors ?
A noncompetitive inhibitor
•Does not resemble the substrate
•Does not bind to the active site, binds to a different site on the enzyme
•Binds to free enzyme (E) or enzyme substrate complex (ES)
•E + S -> ES + I -> ESI OR E + I ->EI + S -> ESI
•Inhibitor does not affect binding of substrate to active site
•Binding of inhibitor brings about conformational change, does not allow reaction to proceed, lowers reaction velocity
•Lowers the Vmax of the reaction
•Km remains unchanged
•Not reversed by increasing substrate concentration
- Lead inhibits ferrochelatase, ALA dehydratase in heme synthesis
- Fluoride inhibits enolase (in glycolysis)
- Cyanide inhibits cytochrome oxidase (in electron transport chain)
What is Mechanism-based enzyme inhibition (‘Suicide’ inhibitors)
- Type of irreversible inhibition
- Mimic or participate in an intermediate step of the catalytic reaction
- May include
- Substrate analogs that bind to enzyme at active site ® converted to more effective inhibitor ® Irreversibly inactivate the enzyme
- Compounds which covalently modify catalytic residues in the active site
For the following drugs name the enzyme involved and the drugs signifcance
Aspirin, Disulfiram, Penicillin (transition state analog), Acyclovir, Allopurinol, 5-Fluorouracil , Di-isopropyl fluorophosphate (DIFP; Organophosphorus compound)
How is ethanol used to treat methanol poisoning?
- Methanol - a solvent in paints & anti-freezes
- May be consumed by accident or may be present as a contaminant in illegally prepared liquor
- Methanol -> formaldehyde -> formic acid
alcohol DH aldehyde DH
- Accumulation of formate leads to metabolic acidosis, tissue hypoxia (may cause blindness, coma & death)
- Can be treated in early stages with ethanol
- Alcohol DH has greater affinity for ethanol; preferentially oxidizes ethanol rather than methanol
What are the two tyes of regulation of enzyme activity?
•Most enzymes operate near equilibrium conditions -> change in substrate concentration -> affects rates of most enzymes.
•Some enzymes have specialized regulatory functions –catalyze rate limiting reactions -> regulated in 2 ways
1.Control of enzyme activity (existing)
->Change in enzyme activity- seconds to minutes (short-term)
•Allosteric regulation
•Covalent modification
2. Control of enzyme availability (new)
- > Change in enzyme activity- hours to days (long-term)
* -Induction and repression *
What are alloseric enzymes? what are they regulated by? What does allosteric enzymes do to the Km and Vmax of the enzyme?
Allosteric enzymes
- contain more than one subunit (polypeptide chain)
- are the regulatory enzymes of metabolic pathways
- are usually the first enzyme (or one of the first few enzymes) of the pathway
- are regulated by molecules known as effectors
- effectors bind to the enzyme non-covalently at a site called allosteric site (different from the active site)
- Bring about conformational change in active site ® alter the Km or Vmax of the enzyme
Allosteric enzymes cont’d
What are positive effector? What are negative effectors? What arehomotropic effectors vs .heterotropic effectors?
- *Positive effectors (allosteric activators**) – activate the enzyme
- *Negative effectors (allosteric inhibitors)** – inhibit the enzyme
Homotropic effectors
When the substrate of the enzyme itself serves as an effector (positive)
Substrate binds to one subunit, increases the rate of substrate binding to other subunits
– co-operative binding (sigmoid curve for v0 vs [S]) eg. hemoglobin
Heterotropic effectors
Effector is different from the substrate
May be positive or negative effector
ALLOSTERIC ENZYMES SHOW SIGMOID CURVE !!
Give an example of allosteric enzyme regulation(3)
1. •Aspartate transcarbamoylase (ATCase) involved in pyrimidine synthesis
•Activated by ATP, inhibited by CTP
2.Isocitrate dehydrogenase (TCA cycle)
Negative effector – ATP
Positive effector – ADP
3. Phosphofructokinase-1 (glycolysis)
Negative effector – citrate
Positive effector – fructose 2,6-bisphosphate
What are the two types of inhibitionand one type of activation seen in allosteric enzymes
Product of an enzyme inhibits the enzyme – product inhibition
A -\>B (accumulation of B, inhibits enzyme)
End product of a pathway inhibiting the key enzyme – feedback inhibition
An earlier intermediate in a pathway, activating the enzyme – feedforward activation
What is covalent modification?
Exlpain what protein kinases do in comparison to phosphoprotein phosphatases .
give an example
-Regulation of an enzyme by addition or removal of a group to the enzyme through a covalent bond
-Addition or removal of phosphate group to/from specific amino acid residues (ser, tyr) in the enzyme phosphorylation or dephosphorylation
-Phosphorylation catalyzed by protein kinases
-Dephosphorylation catalyzed by phosphoprotein phosphatases
-May activate/inactivate the enzyme
-Eg: glycogen phosphorylase – active in phosphorylated state; inactive in dephosphorylated state
When are these enzymes active and inactive?
Acetyl-CoA carboxylase
Glycogen synthase
Pyruvate dehydrogenase
HMG-CoA reductase
Glycogen phosphorylase
Hormone sensitive lipase
Phosphorylase b kinase
Control of enzyme avaliablilty: Whaat is induction and repression?
Induction:
Increase in the rate of enzyme biosynthesis/ increasing the transcription
Repression:
Decrease in the rate of enzyme biosynthesis
Achieved by regulation of gene coding for the enzyme
Takes longer (hours to days) to take full effect
Enzymes that are in constant use are not subjected to induction/repression. Individual enzyme efficiency is not changed
Does not change the catalytic efficiency of existing enzyme molecules
Give example of 4 enzymes that are being induced and repressed
Uses of enzymes in diagnosis. What are the two classes which plasma enzymes belong to?
Plasma enzymes belong to 2 classes:
Actively secreted into the blood
Eg: Liver secretes enzymes involved in blood coagulation
- *Enzymes released into the blood due to cell turnover**
- >Always function intracellularly, no functional role in plasma
- >Fairly constant level in plasma
- >Damage or death of the tissue where a particular enzyme is present in high amounts
lead to..
release of that enzyme into blood
lead to...
Can be measured in blood to diagnose disease
What are isoenzymes? How do they differ? (5)
Physically distinct and separable forms of an enzyme that catalyze the same reaction
Isoenzymes differ in
- >Structure (a.a sequence, subunit composition)
- >Location (tissue or subcellular compartment)
- >Stability (eg: heat)
- >Electrophoretic mobility
- >Susceptibility to inhibitors
Knowledge of the tissue distribution of specific isoenzymes- exploited in diagnostic tests
What are the isoenzymes of creatine kinase? How many subunits does CK have? Where is each one present predominantly? How can isoenzymes be seperated?
CK has 2 subunits (dimer)
Subunits may be B (brain) type or M (muscle) type polypeptide chains
Following a myocardial infarction, CK-2 levels in blood
Total CK level is increased after MI & in sk muscle disorders
Isoenzymes can be separated by electrophoresis
After electrophoretic seperation of CK, why is CK-MM the highest? Which CK primarily reflects the total CK?
Because we have more of skeletal muscle than any other muscle.
•Total CK measurement primarily reflects CK-MM
What are the 5 isoenzymes of lactate dehydrogenase and which tissue they are found? Normally which LDH is higher? After MI which LDH is higher? When does total LDH increase?
LDH has 4 subunits (tetramer)
Subunits may be H (heart) type or M (muscle) type
Normally LDH2 > LDH1
After myocardial infarction -> LDH1 > LDH2 (‘flipped’)
Total LDH in hemolytic anemias, liver disease, cancers
Which LDH ismore common after a MI? Which LDH is more common in acute hepatits?
LDH1- MI
LDH5- Acute hepatitis
When is ALP increased?
ALP: Extrahepatic obstruction, bone diseases
In order explain which isoenzyme is released first, second and third
Which one is coming out first?
- Creatine kinase ( more specific)
- Aspartate transaminase
BUT lactate dehydrogenase( non specific) circulates more for a longer time
After an MI when does each isoenzyme CK-MB, LDH-1 and AST rise begin and when does it return to normal?****
What are the non-enzymatic markers for diagnosis of MI?
Non enzyme markers
•Myoglobin
-released from damaged myocardium 1 -3 hr after MI
-First marker to ; sensitive but not specific
•Cardiac troponins: troponin I & T (cTnI, cTnT)- gold standard for MI
inc 4 – 6 hr after MI, peaks 8 – 28 hr, remain elevated upto one week
üMore sensitive & specific for damage to myocardium than CK-MB
Enzyme markers - CK-MB, LDH-1
CK-MB normalizes by the third day, will again if there is a re-infarction; while troponins stay elevated from the first infarction
Name the serum enzyme markers for the following dieases
- MI
- Hepatitis
- Cholestasis (obstructive jaundice In the bile duct)
- Bone disorders
- Prostatic carcinoma
- acute pancreatitis
- Hemolytic anemia, cancers
- Muscle dystrophies
- Obstructive jaundice
MI: CK2, LDH-1, Aspartate transaminase (AST)
Hepatitis: alanine transaminase(ALT) , AST, LDH
Cholestasis( obstructive jaundice in the bile duct): Alkaline phosphatase(ALP). y-glutamyl transferase(GGT) GGT also inc by alcohol intake
What are proenzymes and what is the significance of proenzymes?
Inactive precursors of active enzymes (zymogens)
Significance of proenzymes:
Some enzymes are synthesized in an inactive form in order to
- >protect the tissue of origin from autodigestion
- >to enable their storage so that they may be released only intermittently but rapidly in response to an immediate physiologic (eg. Digestive enzymes) or pathologic need ( eg clotting fators)
Name 5 proenzymes and their active form and each ones function
What are therapeutic applications of enzymes? (4)
1.Streptokinase & urokinase
Lyse intravascular blood clots
Used to treat myocardial infarction
2. Trypsin, chymotrypsin, lipase, amylase
Used to treat indigestion due to pancreatic insufficiency
3. Asparaginase – used as anticancer drug
- Enzyme replacement therapy for lysosomal storage diseases (Eg: for Gaucher disease)
