Download Chapter 8 Enzymes: basic concepts and kinetics

Chapter 8
Enzymes: basic concepts and kinetics
Light production by enzyme catalysis: energy conversion
Outline:
• enzymes are powerful and specific catalysts
• free energy is a useful thermodynamic function for
understanding enzyme catalyzed reactions
• enzymes accelerates reactions by facilitating the
formation of the transition state
• the Michaelis-Menten model accounts for the
kinetic properties of many enzymes
• enzymes can be inhibited by specific molecules
Enzymes are specific catalysts (8.1)
The peptide bond is thermodynamically unfavorable
but kinetically very stable (half-life 1000 years).
Why?
Reaction catalyzed by a
proteolytic enzyme
Specificity of enzyme catalysis
Trypsin and thrombin
are both proteases.
Trypsin
• Trypsin functions in the
degradation of ingested
proteins
• Thrombin acts only on
one protein: fibrinogen.
Thrombin converts
fibrinogen into fibrin and
fibrin polymeri-zes and
forms a blood clot.
Thrombin
Enzymes are powerful catalysts (8.1)
Why is a reaction that occurs spontaneously
catalyzed by an enzyme?
HCO3- + H+
Reaction catalyzed by carbonic anhydrase
Stimulates
O2 release
from Hb
Fysiologische rol koolzuur anhydrase
pH < 7
pH ~ 7.4
[Figuur 7.22]
Rate enhancement by enzymes
Carbonic anhydrase is one of the fastest enzymes
known. One enzyme molecule can hydrate 106
molecules of CO2 per second.
Many enzymes require cofactors for activity
Apoenzyme + cofactor = holoenzyme
Cofactors that are (small)
organic molecules are called
coenzymes.
cofactor
If coenzymes are tightly
bound, they are called
prosthetic groups.
metalen
If coenzymes are only bound
during catalysis, they are
called cosubstrates.
prosthetische
Metals like Zn, Ni are tightly
bound.
groep
coenzym
cosubstraat
Animatie
De
covalent
gebonden
cofactor,
pyridoxal
Zink,
een covalent
gebonden
cofactor
aan het
fosfaat,
die een prosthetische
enzym (coenzym)
koolzuur anhydrase
groep is van het enzym glycogeen fosforylase
Enzymes can transform energy from one
form into another
Figuur 13-2
• Pomp heeft twee conformaties.
• Een met de ion bindingsplaatsen open voor een kant
van het membraan.
• De andere conformatie is open voor de andere kant.
• Een gekoppelde vrije energie leverende reactie
verschuift het evenwicht tussen de twee conformaties.
Enzymes can transform energy from one
form into another
Structuren van de calcium pomp
Calciumbindingsplaatsen
bereikbaar vanuit het
cytoplasma [Fig 13-3]
Calciumbindingsplaatsen niet
meer aanwezig en open naar de
ander kant van het membraan
[Fig 13-4]
Enzymes can transform energy from one form
into another
The Ca2+ ATPase
uses the energy
of ATP hydrolysis
to transport Ca2+
across the
membrane,
generating a Ca2+
gradient.
Figuur 13-5
Free energy is a useful thermodynamic function
for understanding enzyme catalyzed reactions
(8.2)
The free energy difference of the products minus
that of the substrates of a reaction determine the
direction of the reaction (ΔG).
The reaction: A + B
C+D
[C ].[ D]
ΔG = ΔG + RT ln
[ A].[ B]
0
Free energy is a useful thermodynamic function for
understanding enzyme catalyzed reactions (8.2)
Een reactie kent twee thermodynamische
eigenschappen
• Het vrije energieverschil tussen de producten en de
reactanten bepaalt of een reactie spontaan kan
verlopen (ΔG).
• De vrije energie die nodig om de activeringsenergie
te overwinnen bepaalt hoe snel een reactie verloopt
(ΔGǂ).
[C ].[ D]
ΔG = ΔG + RT ln
[ A].[ B ]
'0
'
At pH 7 add '
[C ].[ D]
ΔG = 0 = ΔG + RT ln
[
A
].[
B
]
0'
'
'
At equilibrium
'0
ΔG = − RT ln K eq
R = gas constant = 8.315 10-3 kJ. mol-1. K-1
T = temperature in K, 25 °C = 298 °K
ln x = 2.303 log x
Together RTlnx = 5.71logx
'
0'
log K eq = ΔG / − 5.71
0
'
[
C
][
D
]
'
− ΔG / 5.71
K eq =
= 10
[ A][ B ]
If Δ G0' = -5.71 kJ.mol-1, the equilibrium shifts a
factor of 10 to the right!!! Hydrogen bonds have
energies ranging from 4 to 20 kJ.mol-1
Calculate the free energy for the isomerization of
DHAP to GAP
At equilibrium the ratio GAP/
DHAP is 0.0475 at 25 °C and
pH 7, calculate the standard
free energy.
• ΔG0' = -2.303 x R x T x log K'eq
= -2.303 x 8.315 x 10-3 x 298
x log (0.0475) = 7.55 kJ mol-1
• The reaction is endergonic under
the standard conditions.
• What will happen when the concentration
DHAP is 2 x 10-4 M and GAP is 3 x 10-6 M?
• ΔG' = 7.55 + 2.303 x RTlog([GAP]/[DHAP])
= 7.55 – 10.42
= -2.87 kJ mol-1
• The reaction is exergonic.
The criterion of spontaneity is
determined by the ΔG and not by the
ΔG0'.
Enzymes accelerate reactions by facilitating the
formation of the transition state (8.3)
v = factor x [s] x
ǂ/RT
-ΔG
e
• The free energy
difference of the
products minus that
of the substrates of
a reaction determine
the direction of the
reaction (ΔG)
• The free energy of activation of a reaction
determines the rate of a reaction (ΔG‡)
• The essence of catalysis is optimizing the structure
for the interactions of the transition state.
• If the transition state is stabilized by 5.71 kJ.mol-1, the
rate increases 10 fold.
Figuur 9.38
en 9.42
Bindingsenergie wordt gebruikt om de DNA dubbelhelix te buigen
waardoor water de fosfodiesterbinding kan bereiken en hydrolyseren
The formation of an enzyme-substrate complex
is the first step in enzymatic catalysis
k2
k1
E+S
ES
k-1
k-2
E+P
The formation of an
enzyme-substrate
complex (ES)
The three dimensional
structure of the catalytic
subunit of protein A kinase.
The inhibitor contains a
pseudosubstrate sequence
Arg-Arg-X(Asn)-Ala(Ser,Thr)-Ile
Properties of the active sites of enzymes
• The active site is a three dimensional cleft or
crevice
• The amino acid residues involved in binding the
substrate(s) are called the catalytic groups
• The active site takes a relative small part of the total
volume of an enzyme
• Substrates are bound to enzymes by multiple weak
interactions
• The specificity of binding depends on the precisely
defined arrangement of atoms in an active site
• Enzymes are flexible and the active site can be
formed by the binding of the substrate (induced fit),
extra free energy when bound water is liberated
Ribbon diagram (A) and a schematic
representation of the primary structure of
lysozyme (B)
The active site can be formed by amino acid residues
from different parts of the polypeptide chain
Substrates are bound by
multiple weak
interactions:
• electrostatic interactions
• hydrogen bonds
• van der Waals forces
• hydrophobic interactions
-CH3
group in
thymine
Hydrogen bonds between
ribonuclease (enzyme) and
the uridine component of its
substrate induce a high
degree of specificity
Three hydrogen bonds ~ 3*-8.4
= - 25.11 kJ.mol-1. Change in
equilibrium about 25000 times
'
K eq = 10
0
'
− ΔG / 5.71
= 10
25.11 / 5.71
= 25808
Enzyme kinetics: the Michaelis-Menten model (8.4)
• The study of the rates of chemical reactions is
called kinetics.
• The study of the rates of enzyme-catalyzed
reactions is called enzyme kinetics.
• A kinetic description of enzyme activity (v) will help
understand how enzyme functions.
Rate of a chemical reaction
S
k
P
rate (v) = - ΔS/Δ T = k x [S]; k = rate constant
Rate of an enzyme catalyzed reaction
The formation of an enzyme-substrate complex
is followed by product formation
k1
E+S
k2
ES
k-1
E+P
k-2
The rate the reaction is the rate of product formation
Determination of the rate as a function
of the substrate concentration
k2
k1
E+S
ES
k-1
E+P
k-2
The initial velocity (V0) is determined under steady
state conditions
Enzyme catalysis with formula
k1
E+S
k2
ES
k-1
v = Vmax [S]/([S] + KM)
KM = (k2 + k-1)/k1
Vmax = k2 [ET]
Vmax = kcat [ET]
E+P
v = k2[ES]
The significance of KM (KM = (k2 + k-1)/k1)
• KM values vary between 10-7 M en 10-1 M
• KM value is the substrate concentration with half of
the binding sites occupied (half maximal velocity)
• The KM value is an indication of the substrate
concentration in vivo
The significance of kcat
- kcat of an enzyme is the number of substrate
molecules that is converted per second into
product per enzyme molecule under saturating
substrate concentrations
- kcat is also called the turnover number.
Vmax = kcat[ET]
- kcat is a direct measure of the catalytic capacity of
an enzyme under saturating substrate
concentrations
- 1/kcat is time of a complete catalytic cycle.
Most biochemical reactions include multiple
substrates
• Sequential reactions
Ternary complex
Most biochemical reactions include multiple
substrates
• Double-displacement (Ping-Pong) reactions
Allosteric enzymes do not obey Michaelis-Menten
kinetics
M-M kinetics
Allosteric kinetics
Enzymes can be inhibited by specific molecules (8.5)
Distinction between competitive, uncompetitve and
noncompetitive inhibition (reversible inhibition)
Competitive, uncompetitive and
noncompetitive inhibition are kinetically
distinguishable
Competitive
Uncompetitive
Noncompetitive
Chapter 15
Metabolism: Basic Concepts and Design
Outline of chapter 15
• Metabolism is composed of many coupled and
interconnected reactions
• ATP is the universal currency of free energy in
biological systems
• The oxidation of carbon fuels is an important source
of cellular energy (redox reactions)
• Metabolic pathways contain many recurring motifs
(the unifying themes of biochemistry)
Living organisms require a continual input of free
energy for:
• the performance of mechanical work in
muscles and other cellular movements
• active transport of molecules and ions
• the synthesis of macromolecules
The free energy is derived from:
• Sunlight: phototrophs are trapping sunlight in
photosynthesis (conversion of energy-poor
molecules like CO2 into energy-rich
molecules like fatty acids and sugars).
• Oxidation of compounds (foodstuffs):
chemotrophs oxidize (carbon) compounds.
Foodstuffs are generated by phototrophs.
Metabolism is composed of many coupled and
interconnected reactions (15.1)
An example of a metabolic pathway: glycolysis.
The free energy of the overall process must be
negative.
All reactions are catalyzed by enzymes.
The activity of the glycolysis is regulated.
Glucose metabolism in humans
•Glucose is metabolized to pyruvate in 10
linked reactions.
•Under anaerobic conditions pyruvate is
metabolized to lactate (2 ATP).
•Under aerobic conditions pyruvate
oxidized to CO2 and H20 via acetyl CoA
and the TCA cycle and respiratory chain
(30 ATP).
Free energy of metabolites of glycolysis in
red blood cells
(138) = concentration in μM
-
-
-
-
Metabolic pathways can be divided into:
• Catabolic reactions: catabolism: fuels
(carbohydrates, fats)
CO2 + H2O + useful
energy
• Anabolic reactions: anabolism: useful energy +
small molecules
complex molecules
• Some pathways can be either anabolic or catabolic,
depending on the energy conditions of the cell.
They are referred to as amphibolic pathways
De citroenzuurcyclus als amfibole route
De citroenzuurcyclus wordt gebruikt om acetyl-groepen af te
breken (katabolisme), maar dient ook als bron voor biosynthese
(anabolisme).
De omzetting van pyruvaat naar oxaloacetaat is hiervoor vereist.
Een anaplerotische reactie
Een belangrijke reactie in de vorming van glucose uit aminoen ketozuren is de carboxylering van pyruvaat tot
oxaloacetaat. Deze reactie is gekoppeld aan ATP hydrolyse
en wordt gekatalyseerd door het biotine bevattende enzym
pyruvaat carboxylase. De door pyruvaat carboxylase
gekatalyseerde reactie verloopt in drie stappen:
1) HCO3- + ATP
HOCO2-PO32- + ADP
2) Biotine-enzym + HOCO2-PO32-
CO2-biotine-enzym + Pi
3) CO2-biotine-enzym + pyruvaat
oxaloacetaat
biotine-enzym +
Somreactie:
Pyruvaat + HCO3- + ATP
oxaloacetaat + ADP + Pi + H+
• De overall reactie heeft een standaard vrije energie van 0.8
kJ. mol-1.
• De hydrolyse van ATP tot ADP en Pi heeft een standaard
vrije energie van -31.4 kJ. mol-1.
• In de reactiecyclus wordt CO2 geactiveerd. De splitsing van
CO2 van het CO2-biotine-enzym complex heeft een standaard
vrije energie van -19.3 kJ. mol-1. Dit hoog energetisch
intermediair wordt gebruikt om pyruvaat te carboxyleren.
• Welk belangrijk principe wordt door pyruvaat carboxylase
gedemonstreerd?
ATP hydrolysis drives metabolism or can perform
work by shifting the equilibrium of coupled
reactions
A
B
ΔG0' = + 16.7 kJ mol -1
K'eq = [Beq]/[Aeq] = 10 - ΔG0'/5.71 = 1.19 x 10-3 = 1 / 841
At equilibrium 841 molecules of A and 1 molecule B or a
protein in conformatie A or B!!
Coupled with ATP hydrolysis (ΔG0' = -30.6 kcal mol -1)
B + ADP + Pi + H+
A + ATP + H2O
(ΔG0' = 16.7 – 30.6 = -13.9 kJ mol -1)
K'eq = [Beq]/[Aeq] x ([ADP]eq [Pi]eq)/[ATP]eq
= 10 - ΔG0'/5.71 = 2.72 x 102 M-1
The ATP-generating system in the cells maintains the
ATP]/[ADP][Pi] ratio around 500 M-1. With this ratio is the
equilibrium between A and B is shifted further towards excess B.
K'eq x [ATP]cel/([ADP]cel [Pi]cel)= [Beq]/[Aeq]
[Beq]/[Aeq] = 2.72 x 102 x 500 = 1.36 x 105
Thus by coupling with ATP hydrolysis the ratio [A]/[B] shifts from
1
A 841
to
=
B
1 136000
Why is ATP an energy-rich molecule?
ΔG0’ = - 30.6 kJ/mol
• ATP + H2O
ADP + Pi + H+
:
• ADP + H2O
AMP + Pi + H+
• AMP + H2O
adenosine + Pi + H+ : ΔG0’ = -14.3 kJ/mol
:
ΔG0’ = - 30.6 kJ/mol
The structural basis of the high phosphoryl
transfer potential of ATP
•Resonance stabilization
•Electrostatic repulsion
•Stabilization of
phosphate by hydration
3x
2x
2x
Free phosphate (4x) has more energetic favorable resonance
structures compared with the terminal phosphate of ATP or ADP
The amount of ATP is limited.
ATP is continuously regenerated
• 100 g ATP in your
body
• In rest 40 kg turnover
in 24 hours. Turnover
3.6 min
• Running: 500 g / min.
Turnover 0.2 min
The ATP-ADP cycle
The oxidation of carbon fuels is the only source of cellular
energy for animals but not for microorganisms (15.3)
(H2 gas)
Free energy of oxidation of single carbon
compounds
In aerobic organisms the electron acceptor in the oxidation
of carbon and hydrogen is O2 and the oxidation products
are CO2 and H2O
Fats are more efficient fuel source than
carbohydrate because carbon is more
reduced
Stages in the
extraction of
cellular energy
from foodstuffs,
mainly reducing
equivalents
8 e- = 3 x NADH,
1 x FADH2
Extraction of free energy in the form of ATP
from fuel molecules
• In catabolism, some ATP is generated (substrate
level phosphorylation), but most of the free energy is
temporary stored in the reducing equivalents
extracted from fuel molecules.
• The reducing equivalents are transferred to NAD+
and FAD. NADH and FADH2 are formed.
• Reducing equivalents are transferred to an electron
transport chain, a respiratory chain.
• Free energy is stored in a proton gradient that
drives the synthesis of ATP.
Oxidation can be coupled directly to ATP synthesis
De overgang van
een C-H naar een
C-OH binding
produceert
58.6 kJ . mol-1
Energy of oxidation
is trapped as a
high phosphoryltransfer-potential
compound and
then used to form
ATP
Electron transport chains generates ion gradients
across membranes providing an important form of
cellular energy that can be coupled to ATP synthesis
NADH, FADH2
The total process is called oxidative phosphorylation
High-energy electrons: redox potentials and
free-energy changes
-
The relation between a
redox potential change
and the free energy
change of a reactions
is:
ΔG = −nFΔE
'
'
ΔG = − nFΔE
0'
'
0
F = 96.49 kJ.V-1.mol-1
Redox
potential
+
Redox potential (ΔE) and free energy (ΔG)
• A 1.14-volt potential difference between
NADH and O2 drives electron transport
through the respiratory chain. This electron
transport is coupled to the formation of a
proton gradient
(ΔG0 = -2 x 96.49 x 1.14 = -220 kJ.mol-1)
• A strong reductant has a negative reduction
potential, a strong oxidizing agent has a
positieve reduction potential
Standard
reduction
potentials
of
biological
important
reactions
The mitochondrial electron transport chain,
bacterial respiratory chains function essential
similar
ATP synthesis from a proton gradient
Comparison between
photosynthesis and
oxidative
phosphorylation
Figuur 19.25
Licht wordt gebruikt om electronen naar een
sterkere reductor over te brengen
Figuur 18.6
Figuur 19.23
Metabolic pathways contain many recurring
motifs (15.3)
• Activated carriers
• Key reactions are reiterated
• Metabolic processes are regulated in
only three principle ways
Activated carriers of electrons for fuel
oxidation
Structures of the
oxidized forms of
nicotinamidederived electron
carriers
NAD+
• R = H: NAD+
• R= PO3 : NADP+
• The nicotinamide
ring of NAD+
accepts a hydrogen
atom and two
electrons, which is
equivalent to a
hydride ion, H-
NADPH is the reductant in biosynthesis,
NADH is used primarily for the generation
of ATP
Flavin adenine dinucleotide is an electron
carrier
• FAD consists of
flavin
mononucleotide
and an AMP unit
• The molecule
accepts 2
electrons and 2
protons
The redox reaction of FAD
Redox reactions and the involved redox
carriers (NAD(P)+)
H- (hydride) transfer
• The redox reaction catalyzed by NAD+ dependent
redox enzymes.
• NAD+ always functions as coenzyme (cosubstrate)
Redox reactions and the involved redox
carriers (FAD)
H (hydrogen) transfer
• The redox reaction catalyzed by FAD dependent
redox enzymes
• FAD is always bound to the enzyme (prosthetic
group)
Coenzyme A is the carrier for activated acyl
groups
CoA
ΔG0' of hydrolysis is -30.6 kJ mol-1
Carriers used in metabolism
The activated carriers are (kinetically) stable
Key reactions are reiterated throughout
metabolism
The six fundamental reactions types are the
basis of all reactions of metabolism
Oxidation-reduction reactions
Ligation reactions form bonds by using the
free energy from ATP hydrolysis
Isomerization reactions
Group-transfer reactions
Hydrolytic reactions
The hydrolysis of a peptide bond
Lyases: enzymes that catalyze the addition
or the removal of functional groups to/from
double bonds or the cleavage involving
electron rearrangement
rearrangements
Metabolic processes are regulated in three
different principle ways
• The amount of enzymes
– rate of transcription
• The catalytic activity of the enzymes
–
–
–
–
reversible allosteric control
feed back inhibition
reversible covalent modification
hormones coordinate metabolic relations between different
tissues often via reversible covalent modification of key
enzymes
• The accessibility of substrates
– controlling the flux of substrates from one compartment to
another (e.g. cytosol to mitochondria).
Biosynthetic and degradative pathways are
almost always distinct for energetic reasons
[ ATP ] + 12 [ ADP ]
Regulation by the energy charge =
[ ATP ] + [ ADP ] + [ AMP ]
The evolution of metabolic pathways
• Why do activated
carriers such as
ATP, NADH,
FADH2 and
coenzyme A
contain adenosine
diphosphate units?
• Binding to a uracil
unit in a niche of
an RNA enzyme
(ribozyme) in the
RNA world
• In the protein world, the carrier function could be
continued without any adaptation