Medical biochemistry and pathophysiology (5052MBP12Y)
Instelling
Universiteit Van Amsterdam (UvA)
Boek
Biochemistry
Summary of all the lectures for the first part of the course Medical Biochemistry and Pathophysiology (biomedical sciences, year 3). With informative pictures and additional information from the book Biochemistry (Berg)
TEST BANK FOR BIOCHEMISTRY, 7TH EDITION: BY JEREMY M. BERG
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Universiteit van Amsterdam (UvA)
Biomedische wetenschappen
Medical biochemistry and pathophysiology (5052MBP12Y)
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Voorbeeld van de inhoud
Enzymes & enzyme inhibitors
Basic concepts and enzyme kinetics Chapter 8
A reaction will occur spontaneously if the ΔG<0 → so if the Gibbs free energy change of the reaction
is negative – meaning that the reaction is exergonic – energy is released
If ΔG>0, the reaction is endergonic and energy is required for the reaction to occur in that direction
Enzyme kinetics
The reaction between an
enzyme and a substrate is
described by two parameters:
I. The binding/interaction between the substrate and enzyme
The enzyme binds the substrate via the substrate-binding site
The Km is the substrate concentration at which the reaction rate is half the maximum rate
(1/2 Vmax) → If the Km is low, the reaction is already at half-maximum at a low substrate
concentration, which means that the enzyme has a high affinity for the substrate and will
already bind the substrate when it is present in low concentrations
II. The conversion of the substrate to the product – the catalysis
The Vmax of the reaction represents the maximum rate of the reaction – the rate of the
reaction at abundant/saturating substrate concentrations
The Kcat (or turnover-number or k2 in the picture above) measures how efficiently the
enzyme converts the substrate into the product – it is the amount of reactions that is
catalyzed per enzyme per second
𝑉𝑚𝑎𝑥 ∙[𝑆]
The rate of the reaction for Michaelis-Menten kinetics is as follows: 𝑣 = 𝐾𝑚+[𝑆]
- If the substrate concentration [S] is very high the denominator of the equation will be equal to [S]
[𝑆]
(the Km will be so small in comparison that it is negligible) → 𝑣 = 𝑉𝑚𝑎𝑥 ∙ [𝑆] = 𝑉𝑚𝑎𝑥 ∙ 1 = 𝑉𝑚𝑎𝑥
- If [S] is (almost) equal to zero the reaction rate will be close to zero as well
[𝑆] 1 1
- if [𝑆] = 𝐾𝑚 → 𝑣 = 𝑉𝑚𝑎𝑥 2[𝑆] = 𝑉𝑚𝑎𝑥 ∙ 2 = 2
𝑉𝑚𝑎𝑥
Inhibitors of Michaelis-Menten enzymes
Inhibitors that bind enzymes reversibly:
I. Competitive inhibitors
Competitive inhibitors bind the enzyme on the substrate-binding site – the structure of the
inhibitor is similar to that of the substrate/transition-state of the substrate, thus the enzyme
has a higher affinity for the inhibitor than for the substrate → this type of inhibition can be
overcome by a high substrate concentration
This type of inhibition changes the affinity of
the enzyme for the substrate
→ Km ↑ , Vmax unchanged
→ Ibuprofen, statins, transition-state analogs
1
, II. Non-competitive inhibitors
Non-competitive inhibitors bind the enzyme at allosteric sites – sites other than the
enzyme’s active site → this will cause a conformational change and the enzyme will go from
the relaxed state (R-state) to the tense
state (T-state)
The catalytic activity of the enzyme will
decrease with this type of inhibition
→ Vmax ↓ , Km unchanged
→ Echinocandins
Inhibitors that bind enzymes irreversibly:
Irreversible inhibitors covalently bind the enzyme, thus eliminating activity of the enzyme →
because fewer enzymes are active the Vmax is decreased
→ Aspirin
Suicide inhibitors/Mechanism-based inhibitors are a group of irreversible inhibitors that form a
covalent bond with the enzyme. The inhibitor will first bind reversibly. The enzyme will try to catalyze
the inhibitor, however when an intermediate is formed it doesn’t leave the enzyme like the
substrate’s intermediate would (catalytic activity of the enzyme is needed for covalent binding)
→ Penicillin
Allosteric inhibitors
All non-competitive inhibitors are allosteric inhibitors, but not all allosteric inhibitors are non-
competitive
Allosteric inhibitors can inhibit enzyme activity → the substrate can still bind, but the enzyme cannot
catalyze the reaction (non-competitive)
Or they can prevent the substrate from binding (competitive)
Suicide inhibitors
Suicide inhibitors first bind the enzyme reversibly (non-covalent bond) in the same way the substrate
would bind the enzyme → a chemically-reactive intermediate will be generated by the enzyme’s
catalytic mechanism → the intermediate will inactivate the enzyme by covalent modifications
- Thus it is actually the enzyme that commits suicide, because it recognizes the wrong substrate
N,N-dimethylpropargylamine (MAO) is a suicide inhibitor for the enzyme monoamine oxidase
- Monoamine oxidase catalyzes the
oxidative deamination of serotonin and
epinephrine
The flavin prosthetic group of
monoamine oxidase also oxidizes MAO,
which will then covalently bind this
prosthetic group → enzyme irreversibly
inactivated
(In the picture MAO is shown in purple)
2
,The family of CYP enzymes
Cytochrome P450 (CYP) is a protein in the ER and cytoplasm that
transfers electrons, using heme as its prosthetic group
During electron transport the iron ion of the heme group alternates
between the reduced (2+) and oxidized (3+) state
2 groups of CYP enzymes
- those that metabolize xenobiotics (family 2 and 3)
- those that participate in key biosynthesis pathways
biosynthesis of sterols, steroid hormones and vitamin D
CYP enzymes oxidize their substrate – NADPH functions as co-enzyme in this reactions and O2 as co-
substrate
𝑅 − 𝐻 + 𝑂2 + 𝑁𝐴𝐷𝑃𝐻 + 𝐻 + → 𝑅 − 𝑂𝐻 + 𝐻2 𝑂 + 𝑁𝐴𝐷𝑃+
Substrate Co-enzyme
will be will be
oxidized oxidized
Co-substrate
will be reduced
CYP enzymes are mono-oxygenases
NADPH transfers its 2 electrons to a
flavoprotein which transfers these one at a time
to adrenodoxin
The substrate RH will bind the CYP enzyme →
Adrenodoxin donates an electron – heme ion
becomes reduced (2+) → O2 binds to Fe2+ →
Adrenodoxin donates a second electron → the
bonds between the oxygen atoms of O2 is
cleaved – one of the oxygen atoms is released
as water, the other forms a Fe4+=O intermediate
→ the hydroxylated product is formed and the
iron goes back to the Fe3+ state
3
, Drug development
Chapter 36
How are new drugs developed?
Two approaches to drug discovery
- Before: you have a compound → discover what the physiological effect of the compound is → find
a target of the compound
- Now: determine what you want to target → make a compound specific for this target → find out
what effect the compound has (desired effect accomplished? Side-effects?)
Penicillin
Approach nr. 1 → accidentally discovered that bacteria are a target for penicillin
Sir Alexander Fleming discovered penicillin →
discovery by serendipity – combination of
intelligence and fortunate coincidence
Penicillin is a suicide inhibitor
The enzyme DD-transpeptidase catalyzes formation
of peptidoglycans crosslinks via small peptides
Penicillin first reversibly binds DD-transpeptidase →
the enzyme will try to catalyze penicillin in the same way it
does the substrate, however penicillin has a β-lactam ring
which forces the enzyme to bind penicillin instead of release it
→ irreversible inactivation of the enzyme
The β-lactam ring looks similar to the transition-state of the substrate
- the transition-state of the substrate (and thus penicillin) will bind more tightly to the
enzyme than the substrate itself and the activation energy will be decreased
When peptidoglycans can’t crosslink the bacterial cell wall will weaken – osmotic pressure won’t be
compensated and water will enter the bacteria → explosion/cytolysis
Gram-positive bacteria have lots of peptidoglycans in their membranes → cytolysis
Gram-negative bacteria don’t have a lot of peptidoglycans so the effect will only be marginal
4
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