Download Chapter 8 Enzymes: Basic Concepts and Kinetics

Chapter 8
Enzymes:
Basic Concepts and Kinetics
Luminescent jellyfish
Enzymes : Basic Concepts and
Kinetics
• Enzymes : the catalysis of biological
systems.
• Catalysis takes place at a particular site on
the enzyme (= ACTIVE SITE)
• Nearly all known enzymes are “proteins”
8.1 Enzymes are powerful and highly specific catalysts
-Enzymes accelerate reactions by factors of as much as
million or more.
-Most reactions in biological systems do not take place in the
absence of enzymes.
-One of the fastest enzymes known is carbonic anhydrase
(hydrate 106 molecules of CO2 per sec.)
<Proteolytic enzymes>
-In vivo, these enzymes catalyze proteolysis, the hydrolysis
of a peptide bond.
- In vitro, proteolytic enzymes also catalyze a different
but related reaction(=hyrdolysis of an ester bond)
-Trypsin : digestive enzyme.
Quite specific and catalyzes the
splitting of peptide bonds only
on the carboxyl side of lysine
and arginine residues.
-Thrombin : participates in
blood clotting. More specific
then trypsin. Catalyzes the
hydrolysis of Agr-Gly bond.
Papain!
8.1.1 Many enzymes require cofactors for activity
-The catalytic activity of
enzymes depends on
the small molecules
termed cofactors.
-Apoenzyme : without
its cofactor.
-Holoenzyme :
complete, catalytically
active enzyme.
-Cofactors can be divided into two group : metals and small
organic molecules.
Apoenzyme +cofactor = holoenzyme
8.2 Free energy is a useful thermodynamic function
for understanding enzymes
• 8.2.1 The free-energy change provides information about
the spontaneity but not the rate of a reaction
- A reaction can occur spontaneously only if ΔG is negative.
- A system is at equilibrium and no net change can take place if ΔG is zero.
- A reaction cannot occur spontaneously if ΔG is positive.
- ΔG is independent of the path
- ΔG provide no information about the rate of a reaction (activation
energy ΔGŧ)
• 8.2.2 The standard free-energy change of a reaction is
related to the equilibrium constant
A + B ↔C + D
ΔG = ΔG0 + RT In[C][D]/[A][B]
ΔG0 is the standard free-energy change
R is gas constant
T is the absolute temperature
At equilibrium, ΔG = 0
ΔG0 = -RT In[C][D]/[A][B]
The equilibrium constant under standard conditions,
Keq = [C][D]/[A][B]
ΔG0 = -RT In Keq = -2.303RTlog10Keq
Keq = 10- ΔG0 /2.303RT = 10- ΔG0 /1.36
When Keq = 10, ΔG0 = -1.36kcal/mol
-This reaction takes place in
glycolysis.
-At equilibrium, Keq = 0.0475
-ΔG0 = -2.303RTlog10Keq
= -1.36Xlog10(0.0475)
= +1.80kcal/mol
→ DHAP will not spontaneously
convert to GAP
ΔG?
ΔG = ΔG0 + RT In[C][D]/[A][B]
8.3 Enzymes accelerate reactions by facilitating the formation
of the transition state
Enzyme alter only the reaction rate and not the reaction equilibrium
The amount of product is the same whether or not the enzyme is present
-A chemical reaction of substrate S to form product P goes
through a transition state Sŧ
-Gibbs free energy of activation or activation energy ΔGŧ: between substrate
and the transition state
-Enzymes accelerate reactions
by decreasing ΔGŧ, the free
energy of activation.
-How?
Catalysis in the Enzyme’s Active Site
• The catalytic cycle of an enzyme
1 Substrates enter active site; enzyme
changes shape so its active site
embraces the substrates (induced fit).
Substrates
2 Substrates held in
active site by weak
interactions, such as
hydrogen bonds and
ionic bonds.
3 Active site (and R groups of
its amino acids) can lower EA
and speed up a reaction by
• acting as a template for
substrate orientation,
• stressing the substrates
and stabilizing the
transition state,
• providing a favorable
microenvironment,
• participating directly in the
catalytic reaction.
Enzyme-substrate
complex
6 Active site
Is available for
two new substrate
Mole.
Enzyme
5 Products are
Released.
Figure 8.17
Products
4 Substrates are
Converted into
Products.
8.3.1 The Formation of an enzyme-substrate complex is
the first step in enzymatic catalysis
What is the evidence for the existence of an enzymesubstrate complex?
1. The reaction rate increases
with increasing substrate
concentration until a maximal
velocity is reached; saturation
effect!
2. X-ray crystallography has provided high-resolution
images of substrates and substrate analogs bound to the
active sites of many enzymes.
Cytochrome P450
Time-resolved crystallography; exposure to a pulse of light converts
the substrate analogue to substrate
3. The spectroscopic
characteristics of many
enzymes and substrates
change on formation of an ES
complex.
-Tryptophan synthetase catalyzes the
synthesis of L-tryptophan from Lserine and indole-derivative.
-Pyridoxal phosphate (PLP) prosthetic
group
8.3.2 The active sites of enzymes have some common features
-The active site of an enzyme is the region that bonds the
substrates.
-Generalizations concerning their active site :
1. The active site is a threedimensional cleft formed by
groups that come from
different parts of the
amino acid sequence.
2. The active site takes up a
relatively small part of the
total volume of an enzyme.
What are the roles of the
remaining parts?
3. Active sites are unique
microenvironments; water is
usually excluded. Nonpolar
microenvironment enhances
the binding of substrates as
well as catalysis
4. Substrates are bound to
enzymes by multiple weak
attractions. (~3-12 Kcal/mol)
- Non-covalent interactions :
electrostatic interaction,
hydrogen bonds, van der
Waals forces, and hydrophobic
interaction.
Shape complementarity is
crucial.
ribonuclease
5. The specificity of binding depends on the precisely
defined arrangement of atoms in an active site.
Lock and key model
- A substrate must have a matching shape to fit into the site.
Induced fit model
- Enzyme changes shape on substrate binding. The active site forms a shape
complementary to that of the transition state only after the substrate is
bound
The binding Energy between enzyme and substrate is
important for catalysis
The binding energy: The free energy released on binding
The maximal binding energy is released when the enzyme is
in the transition state because the full complement of
complex is only formed in transition state.
8.4 The Michaelis-Menten model accounts for the kinetic
properties of many enzymes (판서)
Kinetics is the study of reaction rates
- The extent of product formation is determined as a
function of time for a series of substrate concentrations.
-Vo : defined as the number of moles of product
formed per second.
-Km : equal to the substrate concentration at which the
reaction rate is half its maximal value. Vmax/2
V0 = Vmax[S]/Km+[s] Michaelis-Menten equation
1/V0 = Km+[s] /Vmax[S]
1/V0 = Km /Vmax[S] + [s] /Vmax[S]
= Km /Vmax[S] + 1/Vmax Lineweaver-Burk equation
8.4.1 The significance of Km and Vmax values
-
The Km values of the enzymes range widely.
Km provides approximation of substrate concentration in vivo.
For most enzymes, Km lies between 10-1 and 10-7M.
High Km indicates weak binding.
Low Km indicates strong binding.
-The maximal rate, Vmax reveals
the turnover number of an
enzyme.
-Turnover number : the number
of substrate molecules converted
into product by an enzyme
molecule in a unit time when the
enzyme is fully saturated with
substrate. Also called kcat.
- Most enzymes range from 1 to 104 per sec.
8.4.2 Kinetic perfection in enzymatic catalysis : The kcat/Km
criterion
Multienzyme complex to overcome
diffusion using tunnel
-Most enzymes are not saturated with substrate
(In physiological condition, [s] << Km)
-kcat/Km can be used as a measure of catalytic efficiency.
-Diffusion limits the catalytic efficiency. Why? kcat/Km < K1
!
2-16. Multifunctional Enzymes with
Tunnels
Class 3. Some bifunctional enzymes shuttle unstable intermediates
through a tunnel connecting the active site.
; A physical channel allows the product of one reaction to diffuse
through the protein to another active site
Figure2-44.The two active sites of the bifunctional enzyme tryptophan
synthase are linked by an internal channel
2-16. Multifunctional Enzymes with Tunnels
-Carbamoyl phosphate synthetase
-The single-chain protein has three
separate active sites connected by two
tunnels through the interior of the
protein
-The entire journey from first substrate
to final product covers a distance of
nearly 100Å
Ammonia + carboxyphosphate  carbamate
Carbamate + ATP  carbamoyl phosphate + AD
Figure2-45.Three consecutive reactions are catalyzed by the three
active sites of the enzyme carbamoyl phosphate synthetase
- Chymotrypsin clearly has a preference for cleaving
next to bulky, hydrophobic side chains.
8.4.3 Most biochemical reactions include multiple
substrates
-Most reactions in biological systems usually include
two substrates and two products.
-Multiple substrate reactions can be divided into two
classes.
1. Sequential displacement
2. Double displacement
1. Sequential displacement
- Ordered.
Lactate dehydrogenase
①
②
①
②
- The coenzyme always binds first and the lactate is
always released first.
1. Sequential displacement
- Random.
Creatine kinase
①
①
②
②
- The order of addition of substrates and release of
products is random.
2. Double-Displacement(Ping-Pong)
Aspartate
aminotransferase
-One or more products are released before all substrates
bind the enzyme.
-Substituted enzyme intermediate
8.4.4 Allosteric enzymes do not obey MichaelisMenten kinetics
- These enzymes consist of multiple subunits and multiple
active sites.
-Allosteric enzymes often
display sigmoidal plots.
(hyperbolic plots)
-In allosteric enzymes, the
binding of substrate to one
active site can affect the
properties of other active sites
in the same enzyme molecule.
-Cooperative, regulatory
molecules
8.5 Enzymes can be inhibited by specific molecules
-The activity of many enzymes can by inhibited by the binding of specific small
molecules and ions.
-Competitive inhibitor
Enzyme can bind substrate
or inhibitor but not both.
Inhibitor resembles substrate.
Uncompetitive inhibitor
Inhibitor and substrate bind to different binding site.
But inhibitor bind to only to the enzyme-substrate complex
-Noncompetitive inhibitor
Inhibitor and substrate bind to different binding site.
- Irreversible inhibitor : dissociates very slowly from its
target enzyme. Tightly bound to the enzyme, either
covalently or noncovalently. Important drug.(ex. Penicillin,
Aspirin)
- Reversible inhibitor : rapid dissociation of the enzyme –
inhibitor complex.
- Methotrexate : structural analog of
tetrahydrofolate. Competitive
inhibitor. Used to treat cancer.
(DNA thmine systhesis block!)
8.5.1 Competitive and noncompetitive inhibition are
kinetically distinguishable
-In competitive inhibition,
the inhibitor competes with
the substrate for the active
site.
-Can be overcome by a
sufficiently high
concentration of substrate.
-Inhibitor increase the Km
value.
8.5.1 Competitive and noncompetitive inhibition are
kinetically distinguishable
In uncompetitive inhibition.
Inhibitor bind only to the ES
complex.
But ESI complex can not
produce product.
Vmax is decreased.
Km is reduced.
8.5.1 Competitive and noncompetitive inhibition are
kinetically distinguishable
In noncompetitive inhibition.
Substrate can still bind to the
enzyme-inhibitor complex.
But complex can not produce
product.
Vmax is decreased.
Km is unchanged.
1/Vmax
-1/Km
1/Vmax
-1/Km
1/Vmax
-1/Km
8.5.2 Irreversible inhibitors can be used to map the active site
Irreversible inhibitors can be divided into three
categories
1.Group-specific reagents
2.Affinity labels or reactive substrate analogs
3.Suicide inhibitors
1. Group-specific reagents
-react with specific R groups of amino acids.
-DIPF modifies only 1 of the 28 serine residues in the
chymotrypsin and also modifies reactive serine residue in
acetylcholinesterase.
1. Group-specific reagents
-react with specific R groups of amino acids.
- Iodoacetamide modifies reactive cystein of enzyme.
2. Reactive substrate analog (Affinity labels)
- Structurally similar to the substrate for the enzyme
that covalently modify active site residues.
-TPCK is a substrate
analog for chymotrypsin.
-TPCK irreversible bind
at the active
site(histidine).
2. Reactive substrate (Affinity labels)
- Structurally similar to the substrate for the enzyme
that covalently modify active site residues.
- Bromoacetol phosphate, an analog of dihydroxyacetone
phosphate, binds at the active site of the enzyme.
3. Suicide inhibitors
- The inhibitor binds to the enzymes as a substrate and catalyzed, then
generates a chemically reactive intermediate that inactivates the enzyme
through covalent modification.
- a suicide inhibitor of monoamine oxidase
3. Suicide inhibitors
- The inhibitor binds to the enzymes as a substrate and
catalyzed, then generates a chemically reactive intermediate
that inactivates the enzyme through covalent modification.
- The drug (-)deprenyl, which is
used to treat Parkinson disease
and depression, is a suicide
inhibitor of monoamine oxidase.
8.5.3 Transition-state analogs are potent inhibitors of
enzymes
Tetrahedral  trigonal
- Compounds resembling the transition state of a
catalyzed reaction should be very effective inhibitors.
(Linus Pauling)
-Transition-state analogs.
8.5.4 Catalytic antibodies demonstrate the importance of
selective binding of the transition state to enzymatic activity
- Antibodies that recognize transition states should function as
catalysts
- Catalytic antibodies can be produced by using transition-state
analogs as antigens
- Ferrochelatase catalyzes the insertion of
iron (Fe2+) to protoporphyrin to produce
heme.
- N-methylmesoporphyrin, a bent prophyrin
that resembles the transition state of the
ferrochelatase catalyzed reaction, was used
to generate an antibody. catalytic antibody
8.5.5 Penisillin irreversible inactivates a key enzyme in
bacterial cell-wall synthesis
- Structure of Penicillin : undergo a variety of
rearrangements. (unstable structure)
-Penicillin was inferred to
interfere with the synthesis
of the bacterial cell wall.
-The cell wall, peptidoglycan,
consists of linear
polysaccharide chains that
are cross-linked by short
peptides.
Yellow : sugar
Red : tetra peptide
Blue : penta glycine
-Formation of cross-links in peptidoglycan by glycopeptide
transpeptidase.
-The terminal amino group of the pentaglycine bridge in
the cell wall attacks the peptide bond between two Dalanine residues to form a cross-link.
- An acyl-enzyme intermediate is formed in the
transpeptidation reaction leading to cross-link formation.
-Penicillin inhibits the cross-linking transpeptidase by the
Trojan horse stratagem.
-Penicillin is welcomed into the active site of the
transpeptidase because it mimics the D-Ala-D-Ala moiety
of the normal substrate.
-On binding to the transpeptidase, the serine residue at
the active site attacks the carbonyl carbon atom of the
lactom ring of the penicillin to from the penicilloyl-serine
derivative.
-Penicillin acts as a suicide inhibitor.
In 1964 the International Union of Biochemistry
established an Enzyme Commission to develop a
nomenclature for enzymes.
Reactions were divided into six major groups numbered
1 through 6 .