macromolecules Flashcards
Ritonavir
AIDS drug
competitive inhibitor for HIV protease, inhibiting its interaction with the substrate
this inhibitor mimics the tetrahedral intermediate that is expected to resemble the enzymes transition state
penicillin
irreversible inhibitor of glycopeptide transferase (which is essential for bacterial cell wall formation; by cross linking peptidoglycan chains during cell wall synthesis)
the reactive beta-lactam ring of penicillin resembles the normal peptide bond substrate of the enzyme; It reacts with Ser (-OH) in the active site and remains bound to it
penicillin is a “suicide inhibitor” as it remains covalently bound to the active site and is eventually degraded with the enzyme)
Sarin
irreversible inhibitor of acetylcholine esterase
it reacts with serine (-OH) on the active site and forms a stable enzyme-sarin complex
Explanation:
It has a highly electronegative F and therefore it readily dissociates to give F-, leaving the rest of the sarin irreversibly bound to the (-OH) Ser on the active site
covalent modification of CDK2
it’s reversible and phosphorylation results in inhibition
CDK2 is a Ser/Thr protein kinase important for cell division
one level of control for CDK2 is the insertion of a phosphate at the Tyr15 in the active site;
this prevents binding of a substrate, ATP, in the active site
Its activated when the phosphate is removed by a phosphatase, cdc25
covalent modification in zymogens
they are activated by proteolytic cleavage by proteases
A rare example of a monomeric allosteric enzyme
-Glucokinase
Explanation:
GK has a binding cleft between its two domains.
the domains reorient dramatically when the substrates bind; the substrates are glucose and ATP;
you can describe three states of the enzyme–>
‘super-open’– when no substrate is bound
‘intermediate’– when glucose is bound
‘closed’– when both glucose and ATP are bound
In the presence of glucose the intermediate state is slowly formed;
upon binding ATP the closed state is quickly formed;
NOW–IMP!
after the reaction the enzyme would go to the ‘super-open state’ UNLESS glucose is present at high concentrations, in which case, the GK just forms the intermediate state again after the closed state–> therefore, it is ready to catalyse a reaction more quickly
A rare example of a monomeric cooperative enzyme.
How does it work?
-Glucokinase
Explanation:
GK has a binding cleft between its two domains.
the domains reorient dramatically when the substrates bind; the substrates are glucose and ATP;
you can describe three states of the enzyme–>
‘super-open’– when no substrate is bound
‘intermediate’– when glucose is bound
‘closed’– when both glucose and ATP are bound
In the presence of glucose the intermediate state is slowly formed;
upon binding ATP the closed state is quickly formed;
NOW–IMP!
after the reaction the enzyme would go to the ‘super-open state’ UNLESS glucose is present at high concentrations, in which case, the GK just forms the intermediate state again after the closed state–> therefore, it is ready to catalyse a reaction more quickly
allosteric regulators of glucokinase?
- allosteric site is 20 A from the active site
- no natural activators known; artificial activators are drugs (have been shown to increase the insulin secretion in diabetic mice)
Homotropic versus heterotropic allostery
Homotropic- substrate binding to active site causes a conformational change from low activity to high activity;
this gives sigmoid kinetics
Heterotropic- there’s an additional allosteric site;
presence of activator stabilises the high activity form;
presence of inhibitor stabilizes the low activity form;
at high activator concentrations the curve becomes hyperbolic (all subunits-high affinity)
at high inhibitor concentrations sigmoid curve is flatted
PFK structure and allostery
Structure:
- PFK is a tetramer
- each monomer has one substrate site and one allosteric site (the allosteric sites are actually positioned at subunit interphases)
Allostery
- PFK has two substrates- F6P and ATP
- F6P binds cooperatively
- ATP acts as an allosteric inhibitor (stabilises T-form)
- AMP acts as an allosteric activator (stabilizes R-form)
note: ATP therefore has binding sites at both the active site and the regulatory site
about glycogen phosphorylase and control of its activity
-dimeric enzyme
-binds substrates cooperatively
-Control is both allosteric and covalent;
Phosphorylase a (high activity) and b(low activity) both exist in T (inactive) and R(active) forms;
-hormonal control by adrenaline and insulin has effects on covalent modification status of the enzyme
Covalent modification:
-phosphorylation at Ser14 converts phosphorylase b into a;
Allosteric modification: (for both a and b)
- ATP and G6P stabilise the T phase and inhibit enzyme activity
- AMP stabilises the R from and increases enzyme activity
what are the most commonly prescribed flu drugs? how do they work? what makes them successful?
Tamiflu and Relenza;
they are inhibitors of an essential influenza enzyme, neuraminidase.
they are successful because:
- high selectivity (don’t affect human enzymes)
- high affinity (requiring low concentrations)
- high bioavailability (remaining in the patient)
About 2 glycoproteins on surface influenza virus
- haemagglutinin and neuraminidase
- to infect a cell, H binds to sialic acid on the surface of cell
- when viral particles are released, the H again binds to the sialic acid on the surface of the cell–> N is important to cut the sialic acid and hence is essential for the release of the mature infectious particles
What was the first attempt at inhibitors for neuraminidase? why was it unsuccessful?
- transition state analogues
- poor selectivity–as, also affected human enzymes
state the 2 modifications made to the transition state analogues (the first attempt at neuraminidase inhibitor) to result in the design of tamiflu!
- a positively charged group added to position 4, to form H-bonds with glutamate side-chains
- parts of the molecule that didn’t interact with the enzymes were replaced to make the molecule more lipophilic (so, cross membrane readily; improved bioavailability)
`Tamiflu provides successful treatment against which influenza strain? which mutations have resulted in resistance among some of these viruses?
H1N1 strain
H274Y mutation
Which strain is bird flu?
how is its response to tamiflu? why?
it has H5N1 strain
it is resistant to tamiflu; the binding pocket is much more open in the H5N1 strain as a result of which the binding of tamiflu is weak
what was viagra initially developed for?
what is its mechanism of action?
- it was initially developed as angina treatment
- it blocks the active site of phosphodiesterase-5(PDE5)
- PDE5 normally breaks down cGMP (a second messenger)
- increased cGMP leads to various downstream signalling, including, relaxation of smooth muscle
Amino acids with aliphatic and hydrophobic side chains (small to big)
Glycine alanine valine leucine, isoleucine ...and proline
amino acids with positively charged side chain
Arginine
lysine
histidine
(there are also basic)
amino acids with negatively charged side chain
Aspartic acid
glutamic acid
Amino acids with polar side chain
polar due to -NH2 group:
- asparagine
- glutamine
polar due to -OH group:
- Serine
- Threonine
- Tyrosine (this is also aromatic)
amino acids with aromatic side chains
phenylalanine
tryptophan
tyrosine
amino acids with sulfur containing side chains
Cysteine
Methionine
What causes scurvy?
- scurvy is a Vit C deficiency which results in collagen breakdown
- the enzyme that makes hydroxyproline from proline needs Fe(II) ions
- Vit C maintains the Fe(II) ions in the reduced state, allowing the enzyme to work
what is a cofactor? what is a coenzyme? state 2 types of coenzymes.
a cofactor provides chemical reactivity that is not found in the amino acid side chains.
cofactors involved in enzyme-catalysed reactions are called coenzymes
the 2 main classes of coenzymes are:
- prosthetic groups (covalently attached to protein)
- cosubstrates (released after reaction to be regenerated)
give 2 common examples of prosthetic groups and their vitamin source
- FMN, FAD- from riboflavin(B2)
- TPP- from Thiamine (B1)
etc
give examples of cosubstrates and their vitamin source
- NAD+/NADP+ - Niacin (B3)
- CoA - Pantothenate (B5)
- tetrahydrofolate- Folic acid (B9)
How many subunits in a potassium channel? how many helices form a monomer?
K+ channel is a tetramer
each subunit has three alpha helices
How many subunits does Lactate dehydrogenase have? How many isoenzymes exist? how are the isoforms distributed among tissues?
LDH has 4 subunits
there are two genetically distinct subunits: A and B
There are 5 different isoenzymes of LDH found in tissues:
4A (heart) 3A,B 2A,2B (lungs) A, 3B (kidneys) 4B (muscle)
describe the structure of a zinc finger
-two His residues (lying 2 amino acids apart) and two Cys residues (lying 3 amino acids apart ) and separated by a length of amino acids
the structure is organised around the two His and the two Cys which bind a Zn (II) ion
Prosthetic group of aminotransferase
B6 (pyridoxal phosphate)
It acts as a temporary parking spot for amine groups
Leupeptin
- inhibitor of trypsin
- competitive
Type of residue selected by serine proteases
chymotrypsin– bulky hydrophobic
trypsin– positively charged
elastase–small
HIV protease
- an Asp protease
- different from humans (as in HIV it has 2 chains that form a dimer)
- the substrate has Phe and Pro residues on either side of the bond to be broken
Proline racemase inhibitor
- pyrolle-2-carboxylate
- a planar intermediate mimic
- when bound to the enzyme a flat pocket can be seen
Saquinavir `
- also a HIV protease inhibitor
- often used with ritonavir
- mimics the tetrahedral intermediate
glycopeptide transpeptidase
- cross links peptidoglycan during cell wall synthesis
- blocked by penicillin
Irreversible inhibitors
penicillin
sarin
