Medical biochemistry and pathophysiology (5052MBP12Y)
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Samenvatting Molecular biology of the Cell - Medical biochemistry and pathophysiology (5052MBP12Y)
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Medical biochemistry and pathophysiology (5052MBP12Y)
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Universiteit Van Amsterdam (UvA)
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Molecular Biology of the Cell 6E - The Problems Book
Summary of chapters from the Molecular Biology of the Cell book on biochemistry. For the course Medical biochemistry and pathophysiology during the third year of studying biomedical sciences at the UvA
Test Bank For Molecular Biology of the Cell The Problems Book 6th Edition by John Wilson; Tim Hunt 9780815344537 Chapter 1-24 Complete Guide.
Summary Molecular Biology of the Cell 2 (MBOC2)
From DNA to RNA: transcription
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Medical biochemistry and pathophysiology (5052MBP12Y)
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NOTES BIOCHEMESTRY
Chapter 8 Enzymes: basic concepts and kinetics
Characteristics of enzyme are catalytic power and specificity. Nearly all known enzymes are proteins.
Proteins can catalyze reaction by stabilizing transition states à can determine which one of several
potential chemical reactions takes place.
The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme. This is
a result of the intricate three-dimensional structure of the enzyme protein.
Conformation selection = substrate will only bind to certain conformations of the enzyme
Some enzymes depend on cofactor for the catalytic activity. Apoenzyme + cofactor = holoenzyme.
1. Metals
2. Small organic molecules à coenzymes
- Tightly bound coenzymes à prosthetic group
- Loosely bound coenzymes are more co-substrates
Gibbs free energy G à determines if reaction can take place (ΔG) and the degree to which an enzyme
accelerates the reaction (determined by energy required to initiate conversion of reactants into
products)
ΔG à determines if reaction happens spontaneously. If negative it is exergonic (spontaneously). If
positive the reaction requires energy input (endergonic).
For A à B ΔG = ΔG0 + RTln([B]/[A])
0
ΔG = standard free energy change à when each reactant is 1 M thus depending on nature reactant.
Can determine ΔG0 by measuring concentrations of reactants and products at equilibrium.
K’eq = equilibrium constant under standard condition à K’eq = e-ΔG0’/2.47
A chemical reaction of substrate S to product P has a transition state that has a higher free energy
than either S or P. Transition state is least stable. To reach this higher energy you need activation
energy, which is lowered by using a catalysator à enzymes thus facilitate the formation of the
transition state.
An enzyme-catalyzed reaction has a maximum velocity because of the formation of discrete ES
complex, at high S concentration all enzymes will filled, forming ES complexes and thus no further
increase in reaction rate is possible.
Catalytic groups = amino acid residues in active site that directly participate in the making and
breaking of bonds
Maximal binding energy (released upon binding with enzyme) is released when enzyme facilitates
the formation of the transition state.
AàP
Rate (V) = the quantity of A that disappears in a specified unit of time. Is related to [A] by a
proportionality constant k (rate constant)
First order reactions à reactions that are directly proportional to the reactant concentration
Bimolecular reactions à reaction including two reactants
Zero order reaction à does not depend on concentration of reactants
,V0 = rate of catalysis à number of moles of product formed per second at beginning reaction (t≈0)
Vmax = when catalytic sites enzyme are saturated with substrate
- Can also indicate turnover number (Kcat) à Kcat = Vmax/[E]T (concentration of active sites)
KM = substrate concentration at which the reaction rate is V max/2. Low value indicates high affinity of
binding, and high value low affinity. A KM value of 4 nM means at a concentration of 4 nM half of the
substrate is bound
- [S] < KM à low activity, very sensitive changes
- [S] > KM à high activity, low sensitivity to change
- [S] ≈ KM à significant activity and still sensitive to change (favorable)
V0 = Vmax*([S]/([S]+KM))
Fraction of active sites filled (fes) à fes = V/Vmax = [S]/([S]+KM)
Kcat/ KM = specificity constant à can be used to compare enzyme’s preferences for substrates
Multi-substrate reactions can be divided into two classes
1. Sequential reactions
- All substrates must bind to enzyme before product is released
- Can be ordered (substrates bind in specific order) or random
o Coenzyme always binds first, and lactate always is released first
2. Double-displacement reactions
- One or more products released before all substrates bind to enzyme
- Existence of substituted enzyme intermediate à enzyme temporarily modified
Allosteric enzymes do not obey Michaelis-Menten. They can bind a substrate to one active site that
changes conformation of other active site. Can be cooperative binding à binding one substrate
facilitates binding other substrate.
Competitive inhibitor = binds instead of the substrate
- Vmax stays the same, but KM increases
Uncompetitive inhibitor = bind only to ES complex
- Lower Vmax and KM à lower concentration S required to form half of the maximal
concentration of ES
Noncompetitive inhibitor = can bind simultaneously with other substrate. Can bind enzyme or ES
complex. Decreases turnover number. Can’t be overcome by increase in [S].
- Lower Vmax, KM stays the same
Irreversible inhibitors
- Group specific reagents; react to specific side chains of amino acids
- Reactive substrate analogs (affinity labels); structurally similar to substrate of enzyme
that covalently bind to active-side residues
- Suicide inhibitors (mechanism based inhibitors); bind to the enzyme as a substrate that
is initially processed by the normal catalytic mechanism. The mechanism generates
chemically reactive intermediate that inactivates the enzyme through covalent
modification. Thus enzyme participates in own inhibition
Transition state analogs à can be very effective inhibitors since it resembles transition state of a
catalyzed reaction
,Chapter 12.4 Proteins carry out most membrane processes
Different type of membranes differ in protein concentrations.
Integral membrane proteins interact extensively with the hydrocarbon chains of membrane lipids,
and they can be released only by agents that compete for these nonpolar interactions
Peripheral membrane proteins are bound to membrane primarily by electrostatic and hydrogen
bond interactions with the head group of lipids, can be disrupted by adding salt or changing pH
Interactions of proteins with membranes
- Proteins can span membrane with alfa helices. Are most common structural motif in
membrane proteins. Most amino acids are nonpolar and very few are charged.
- A channel protein can be formed from beta sheets. Outside is nonpolar. Inside is
hydrophilic and filled with water.
- Embedding part of protein in a membrane can link the protein to the membrane surface.
Is not largely embedded in the membrane. Lies on surface firmly bound to set of alfa
helices with hydrophobic surfaces that extend form the bottom of the protein into the
membrane. Only action of detergents can release protein from membrane à integral
Chapter 24.3 Feedback inhibition regulates amino acid biosynthesis
Committed step = first irreversible reaction in pathway. Enzyme that catalysis this reaction is often
inhibited by the final product of the pathway. Can be catalyzed by two or more isozymes à enzymes
with essentially identical catalytic mechanism but different regulatory properties.
In cumulative inhibition, each inhibitor can reduce the activity of the enzyme, even when other
inhibitors are bound at saturated levels.
Chapter 28 Drug development
Target based screening: Target validation à assay development à library screening à positive hits
Phenotypic screening: phenotypic assay à library screening à positive hits à target identification
Criteria of compounds for drug development
- Drugs must be potent and selective
o Effectiveness determined by strength of interaction drug and target
o EC50 = concentration drug candidate required to elicit 50% of the maximal
biological response
o IC50 = for inhibitors the concentration required to reduce response by 50%
relative to value in absence of inhibitor. For competitive inhibitor it depends on
concentration and KM of S.
o EC50 and IC50 are measures of potency
o Should be selective to reduce side effects
- Drugs must have suitable properties to reach their targets (ADME)
o Administration and absorption
Ability to be absorbed is quantified in terms of oral bioavailability à ratio
of peak concentration of compound given orally to peak concentration of
same dose injected in bloodstream
Partition coefficient = a way to measure tendency of molecule to
dissolve in membranes
, o Distribution
Effective drug will reach target compartment in sufficient quantities
o Metabolism
Oxidation; increase water solubility and introduce functional group.
Often promoted by cytochrome P450 enzymes in the liver
Conjugation; addition of particular groups to xenobiotic compounds.
Commonly added are glutathione, glucuronic acid and sulfate. Make
them more water soluble and easier to be recognized to target excretion
o Excretion
Enterohepatic cycling à compounds recycled from bloodstream into
intestine and back into bloodstream
- Toxicity can limit drug effectiveness
o Mechanic based / on-target toxicity à modulates target molecule too effectively
o Off target toxicity à modulates the properties of proteins that are distinct from
target molecule
o May also be toxic if it modulates activity of protein unrelated to intended family
o May not be toxic itself but byproducts can be
o Toxicity of drug candidate described in terms of the therapeutic index à ratio of
dose of compound required to kill one half of animals (LD50)
Combinatorial chemistry = approach that synthesizes a large number of structural related
compounds that differ from another at only one or a few positions all at once à used for screening
libraries to indicate new drug leads
Key method of this is split pool synthesis à compounds synthesized on small beads and divided into
number or sets (split). Reactions adding the reactants at the first site are run and are isolated by
filtration. The sets are then combined (pooled), mixed and split again. Reactions adding new
reactants are run and beads isolated again. Each bead only has one compound. But by doing this you
create many more different compounds à find one with desired properties.
Chapter 15 Metabolism: basic concepts and design
Catabolism à reactions that generate useful energy
Anabolism à reactions that require energy to proceed
Criteria pathways
1. Individual reactions must be specific
2. The entire set of reactions that constitute a pathway must be thermodynamically favored
The overall free energy change (ΔG0) for a chemically coupled series of reactions is equal to the sum
of the ΔG0 of the individual steps.
The hydrolysis of n ATP changes the equilibrium ratio of a coupled reaction by a factor of 10 8n.
Because ΔG0 depends on the difference of the free energy of the products and reactants, you need to
look at the structure of both. Four factors are important: resonance stabilization, electrostatic
repulsion, increase in entropy and stabilization due to hydration.
Other role of ATP is to maintain protein solubility, thus preventing protein aggregation, since it is a
hydrotrope à hydrophobic and hydrophilic parts.
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