Unit 3
Enzymes. Catalysis and enzyme kinetics.
3.1. Characteristics of biological catalysts. Coenzymes, cofactors, vitamins Enzyme nomenclature and classification 3.2. Enzyme catalysis. Transition state Active site Enzyme-substrate complex Factors involved in enzyme catalysis 3.3. Enzyme kinetics. Steady-state assumption and Michaelis-Menten equation Factors affecting the enzymatic activity Enzymatic inhibition • Reversible inhibition • Irreversible inhibition 3.4. Enzyme regulation. Allosteric behaviour Covalent modification Proteolysis
OUTLINE
What characteristics features define enzymes?
• High catalytic power: ratio of the catalysed rate to the uncatalysed rate of the reaction = 106-1020
• Enzymes are recover after each catalytic cycle.
• High specificity: (even stereospecifivity)
• Regulation
The biological catalysts are: – Proteins (enzymes) – Catalytic RNA (ribozymes)
3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS
• It converts 6x105 molecules per second
• 107 times faster than the uncatalysed reaction
Ejemplos de reacciones catalizadas
• 1011 times faster than the uncatalysed reaction • The specificity depends on the R1 group.
Protease Carbonic anhydrase
3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS
Nonprotein components required for the enzymatic activity: cofactor – Apoenzyme + cofactor = holoenzyme – Two types of cofactors: • Metal ions: Mg2+, Zn2+, Cu2+, Mn2+, ... • Coenzymes: small organic molecules synthesised from vitamins. Prosthetic groups: tightly bound coenzymes Cofactors deficiency promotes some health problems.
COFACTORS, COENZYMES AND VITAMINS
3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS
3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS
COFACTORS, COENZYMES AND VITAMINS
3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS
COFACTORS, COENZYMES AND VITAMINS
Nº Class Reaction Examples
1 Oxidoreductases Oxidation-reduction reactions Glucose oxidase (EC 1.1.3.4)
2 Transferases Transfer of functional groups Hexokinase (EC 2.7.1.2)
3 Hydrolases Hydrolysis reactions Carboxipeptidase A (EC 3.4.17.1)
4 Lyases Addition to double bonds Piruvate decarboxylase (EC 4.1.1.1)
5 Isomerases Isomerisation reactions Malate isomerase (EC 5.2.1.1)
6 Ligases Formation ob bonds (C-C, C-S, C-O and C-N) with ATP cleavage
Piruvate carboxylase (EC 6.4.1.1)
ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS
Traditional Nomenclature urease: urea hydrolysis amylase: starch hydrolysis DNA polymerase: Nucleotides polymerization • Trivial designations (Ambiguity) Systematic Nomenclature (identify the substrate and the reaction) ATP + D-glucose → ADP + D-glucose 6-phosphate ATP: D-hexose 6-phosphotransferase hexokinase (traditional nomenclature)
ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS
Carboxipeptidase A (peptidyl-L-amino acid hydrolase) EC 3.4.17.1 Class: 3 → Hydrolases. Subclass: 4 → peptide bond 17 → metallocarboxypeptidases. Entry number: 1
A series of four number serves to specify a particular enzyme. The numbers are preceded by the letters EC (enzyme commission). First number: class Second number: subclass (electron donors, type of substrate, etc.) Third number: characteristics of the reaction (functional groups, etc.) Fourth number: order of the individual entries
ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS
The conversion of S to P occurs because a fraction of the S molecules has the energy necessary to achieve a reactive condition known as the transition state (S-P intermediate)
Enzymes (catalysts) work by lowering the free energy of activation related to the transition state
A-B + C
A….B….C
A + B-C
Ej. A-B + C A + B-C
Transition state
3.2. ENZYME CATALYSIS
Substrate binds at the active site of the enzyme through relatively weak forces (chymotrypsin)
Specificity Catalytic power
Active site
3.2. ENZYME CATALYSIS
Lock and key theory (Fisher, 1890)
Induced fit theory (Koshland y Neet, 1968)
Enzyme-substrate complex interactions
3.2. ENZYME CATALYSIS
Glucose induced conformational change of hexokinase
D-glucose
(a) Unligaded form of hexoquinase and free glucose
(b) Conformation of hexokinase with glucose bound
Enzyme-substrate complex interactions
3.2. ENZYME CATALYSIS
FACTORS INVOLVED IN ENZYME CATALYSIS
• Proximity and orientation
• Surface phenomena
• Bounds tension
• Presence of reactive groups
3.2. ENZYME CATALYSIS
Proximity and orientation
FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS
FACTORS INVOLVED IN ENZYME CATALYSIS
Bounds tension
3.2. ENZYME CATALYSIS
Mechanisms of catalysis
General acid-base catalysis: proton transference in the transition state (from or towards the substrate)
Covalent catalysis: transitory covalent bond between enzyme and substrate
Metal ion catalysis: it acts as electrophilic catalysts, it promotes redox reactions, it stabilised charges, the polarity of certain bounds can change because of the metals…
Presence of reactive groups FACTORS INVOLVED IN ENZYME CATALYSIS
3.2. ENZYME CATALYSIS
FACTORS INVOLVED IN ENZYME CATALYSIS
3.2. ENZYME CATALYSIS
General acid-base catalysis and covalent catalysis: protease
Presence of reactive groups
FACTORS INVOLVED IN ENZYME CATALYSIS
3.2. ENZYME CATALYSIS
Enolase General acid-base catalysis and metal ion catalysis
FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS
It is the analysis of the velocity (or rate) of a chemical reaction
catalysed by an enzyme, and how the velocities can change on the
basis of environmental parameters modifications.
WHAT DO YOU HAVE TO KNOW? • How the rate of an enzyme-catalysed reaction can be defined in a mathematical way • Velocity units • What is the order of a reaction (first-order reaction/second order reaction?
3.3. ENZYME KINETICS
Hypothetical enzyme catalyzing: SP The rate of the reaction decreased when S is converted into P. Initial velocity: slope of tangent to the line at time 0
The rate of a enzymatic reactions depends on the substrate concentration
3.3. ENZYME KINETICS
3.3. ENZYME KINETICS The rate of a enzymatic reactions depends on the substrate concentration
Michaelis-Menten equation describes a curve known as a rectangular hyperbola
The velocity of the product formation is:
[ES]kv 2=
[ES] depends on: the velocity of ES formation from E + S the velocity of its dissociation to regenerate E+S or to form E + P.
][][][ 211 ESkESkS[E]kdt
d[ES]−−= −
STEADY-STATE ASSUMPTION AND MICHAELIS-MENTEN EQUATION
E + S ES E + P k1
k-1
k2
3.3. ENZYME KINETICS
0 Time
Early stage ES formation
Steady state [ES] is constant
Steady-state Under experimental conditions [S]>>>[E]. The [ES] quickly reaches a constant value in such dynamic system, and remains constant until complete P formation: Steady State assumption
3.3. ENZYME KINETICS
][][][ ESEE T +=
])[(]][[][][ 2111 ESkkSESkSEk T +=− −
KM, Michaelis constant
])[][(][][ 2111 ESkkSkSEk T ++= −
211
1
][][][][kkSk
SEkES T
++=
−
121 /)(][][][][
kkkSSEES T
++=
−
M
T
KSSEES
+=
][][][][
M
T
KSSEkv
+=
][][][2
Maximal velocity is obtained when the enzyme is saturated: [E]T=[ES]
T[E]kV 2max =
[ES]kv 2=
MKSSVv
+=
][][max
Michaelis-Menten Equation
1
21
kkkK M
+= −
Steady-state
3.3. ENZYME KINETICS
] [ ] [ ] ][ [ , 0 ] [ 2 1 1 ES k ES k S E k
dt ES d + = = − so
3.3. ENZYME KINETICS
What does KM mean?
1
21
kkkK M
+= −
MKSSVv
+=
][][max When [S]=KM, v=Vmax/2
KM is the substrate concentration that gives a velocity equal to one—half the maximal velocity. Units of molarity. It indicates how efficient in an enzyme selecting substrates (specificity) Usually KM is used as a parameter to estimate the affinity of an enzyme for their substrates. KM is similar to the ES dissociation constant when k2<<k-1.
][]][[
1
1
ESSE
kkK M =≈ −
E + S ES E + P k1
k-1
k2
3.3. ENZYME KINETICS
3.3. ENZYME KINETICS The rate of a enzymatic reactions depends on the substrate concentration
Michaelis-Menten
Turnover number, Kcat
Tcat E
Vk][
max=Kcat of an enzyme is a measure of its maximal catalytic activity. It represents the kinetic efficiency of the enzyme
In the reaction kcat = k2
Kcat: turnover number: number of substrate molecules converted into product per enzyme molecule per unit time, when the enzyme is saturated with substrate
First order velocity constant. Units: s-1
E + S ES E + P k1
k-1
k2
3.3. ENZYME KINETICS
3.3. ENZYME KINETICS Turnover number, Kcat
kcat/KM defines the catalytic efficiency of an enzyme It provides information about two combined facts: substrate binding and catalysis (substrate conversion into product).
][][ SEKkv T
M
cat=When [S]<<KM,
Kcat/Km is the velocity constant of the E +S conversion into E + P. Second order constant. Units: M-1s-1
The catalytic efficiency of an enzyme cannot exceed the diffusion-controlled rate of combination of E and S to form ES.
3.3. ENZYME KINETICS
Experimental determination of KM and Vmax
Several rearrangements of the Michaelis-Menten equation transform it into a straight-line equation: Lineweaver-Burk double-reciprocal plot:
maxmax
1][
11VSV
Kv
M +=
3.3. ENZYME KINETICS
Factors affecting the enzymatic activity
Enzyme concentration -Enzymatic activity international unit (U): quantity of enzyme able to transform 1.0 µmol substrate per minute at 25ºC (under optimal conditions)
- Specific enzymatic activity (U/mg): number of enzymatic unit per mg of purified protein. It indicates how pure the enzyme is.
Balls: they represent proteins Red balls: enzyme molecules Both cylinders: same activity units Right cylinder shows higher specific activity than the left cylinder
3.3. ENZYME KINETICS
Temperature The rates of enzyme-catalysed reactions generally increase with increasing temperature. However, at high temperatures the activity declines because of the thermal denaturation of the protein structure.
pH Enzymes in general are active only over a limited pH range, and most have a particular pH at which their catalytic activity is optimal. pH changes can modify side chain, prosthetic groups and substrate charges, and consequently, the activity of the enzyme.
Factors affecting the enzymatic activity
3.3. ENZYME KINETICS
Enzymatic inhibition • Inhibition: velocity of an enzymatic reaction is decreased or inhibited by some agent (inhibitors) – Irreversible • Inhibitor causes stable, covalent alterations in the enzyme – Examples: » Ampicillin: causes covalent modification of a transpeptidase catalysing the synthesis of the bacterial cellular wall » Aspirin: causes covalent modification in a cyclooxygenase involved in inflammation – Reversible • Inhibitor interact with the enzyme through noncovalent association/dissociation reactions.
3.3. ENZYME KINETICS
][]][[
EIIEKI =
IKI ][1+=α
][][SK
SVvm +
=α
REVERSIBLE INHIBITION
The inhibitor binds reversibly to the enzyme at the same site as substrate. The inhibitor resemble S structurally.
S-binding and I-binding are mutually exclusive, competitive processes.
The inhibition is blocked when the substrate concentration increases.
Kmapp increases and V is unaffected
Competitive Inhibition
mappm KK α=
][][1
][
SKIK
SVv
Im +
+
=
3.3. ENZYME KINETICS
Competitive Inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS
IKI ][1+=α
][
][
SK
SVv
m += α
][]][[
EIIEK I =
Noncompetitive inhibition
Inhibitor interacts with both E and ES.
The inhibition is not blocked when the substrate concentration increases.
Vapp decreases and Km is unaffected
′= II KK
][]][[
EIIEK I =
αVVapp =
][][1][1
][
SKI
KIK
SVv
IIm
++
+
=
REVERSIBLE INHIBITION 3.3. ENZYME KINETICS
mK1
−
mK1
−
Noncompetitive inhibition
REVERSIBLE INHIBITION 3.3. ENZYME KINETICS
IKI′
+=][1α
][
][
SKSV
vm +
=
α
α
][]][[
ESIIESKI =′
Inhibitor only combines with ES
It does not bind in the active site.
Vapp and Kmapp decrease
][][1
][
SK
IK
SVv
I
m
′++
=
αVVapp =
αm
appmKK =
Uncompetitive inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS
Uncompetitive inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS
Chymotrypsin inhibition by diisopropylfluorophosphate (DIFP)
Ciclooxigenase inhibition by aspirin
IRREVERSIBLE INHIBITION 3.3. ENZYME KINETICS
Living systems must regulate the enzymatic catalytic activity to: - Coordinate metabolic processes - Promote adaptations to environmental changes - Growth and complete the living cycle in the correct way
Two mechanisms of regulation: 1.- Control of the enzyme availability 2.- Control of the enzymatic activity, by means of modifications of the conformation or structure
3.4. ENZYME REGULATION
Allosteric enzyme: Oligomeric organization (more than one active site and more than one effector-binding site) The regulatory effects exerted on the enzyme’s activity are achieved by conformational changes occurring in the protein when effector metabolites bind Conformational states for a protein (monomer): Taut state (T): Low substrate affinity Relaxed state (R) : High substrate affinity
ALLOSTERIC REGULATION
3.4. ENZYME REGULATION
Homotropic effect: The ligand-induced conformational change in one subunit can affect the adjoining subunit: Cooperativity Usually, it is positive regulation No Michaelis-Menten kinetics Sigmoidal curves
ALLOSTERIC REGULATION
3.4. ENZYME REGULATION
Heterotropic effect: The effectors do not bind in the active site Activator: R state is stabilised Inhibitors: T state is stabilised
ALLOSTERIC REGULATION
3.4. ENZYME REGULATION
Aspartate carbamoyltransferase: allosteric enzyme
As product accumulates, the rate of the enzymatic reaction decreases (negative effect)
Feedback inhibition 3.4. ENZYME REGULATION
Aspartate carbamoyltransferase: allosteric enzyme
COVALENT MODIFICATION
3.4. ENZYME REGULATION
Most of the covalent modification involved in enzyme activity regulation are phosphorylations. One or more than one phosphorylation site Protein kinases: They act in covalent modifications by attaching a phosphoryl moiety to target proteins Phosphoprotein phosphatases: They catalyse the removal of phosphate groups.
COVALENT MODIFICATION
3.4. ENZYME REGULATION
Glucogen phosphorylase (adrenalina)
COVALENT MODIFICATION
3.4. ENZYME REGULATION
Some proteins are synthesized as inactive precursors, called
zymogens or proenzymes, that acquire full activity only upon
specific proteolytic cleavage of one or several of their peptide bonds
It is not energy dependent The peptide bond cleavage is irreversible Examples Digestive enzymes Blood clotting Peptidic hormone (insulin) Collagen Caspases: apoptosis
3.4. ENZYME REGULATION
PROTEOLYSIS
Trypsin cleaves the peptide bond joining Arg15 - Ile16
Chymotrypsin π is an enzymatically active form that acts upon other Chymotrypsin π molecules, excising two peptides. The end product is the mature protease Chymotrypsin α, in which the three peptide chains remain together because they are linked by two disulfide bonds
PROTEOLYSIS COVALENT MODIFICATION
3.4. ENZYME REGULATION