Enzymes
Why Biocatalysis?
•
•
•
•
Higher reaction rates
Greater reaction specificity
Milder reaction conditions
Capacity for regulation
COO
-
COO
NH2
O
OH
COO
-
O
COO
OH
COO
COO
Chorismate
mutase
COO
OOC
O
NH2
OH
-
• Metabolites have
many potential
pathways of
decomposition
• Enzymes make the
desired one most
favorable
Rate enhancement by selected enzymes
Enzymatic Substrate Selectivity
OH
H
-
H
+
NH3
OOC
-
+
NH3
No binding
H
-
OOC
+
NH3
OOC
OH
HO
OH
H
H
H
NH
CH3
Example: Phenylalanine hydroxylase
Binding but no reaction
Three point attachment
of symmetrical substrate
to an asymmetric
binding substrate site
Even a symmetrical
chemical substance can
be selectively bound and
treated by an enzyme
Enzyme Classification and
Nomenclature
• Enzymes are classified on the basis of the types of
reactions that they catalyse
• Trivial names: with suffix -in (historical - trypsin, pepsin, etc)
• Most named for substrates & for reactions, with suffix “ase”
(ATPase breaks down ATP, ATP synthase makes ATP,
lactase, alcohol dehydrogenase ...)
• 1964, systematic classification & nomenclature of enzymes
developed by the Enzyme Commission (EC)
1964 - Systematic classification &
nomenclature of enzymes developed by
the Enzyme Commission (EC):
• Substrate (donor) : (acceptor or cofactor, type of group,
type of isomerization) type of reaction
• The Enzyme Commission number (EC number):
• numerical classification scheme for enzymes, based on the chemical
reactions they catalyze.
• Every EC number is associated with a recommended name for the
respective enzyme.
International classification of enzymes
Group
EC 1
EC 2
EC 3
EC 4
EC 5
EC 6
Reaction
catalyzed
Examples
with trivial
name
Typical reaction
Oxidoreductases
To catalyze oxidation/reduction
reactions; transfer of H and O atoms
or electrons from one substance to
another
AH + B → A + BH (reduced)
A + O → AO (oxidized)
Dehydrogenase,
oxidase
Transferases
Transfer of a functional group from
one substance to another. The group
may be methyl-, acyl-, amino- or
phosphate group
AB + C → A + BC
Transaminase,
kinase
Hydrolases
Formation of two products from a
substrate by hydrolysis
AB + H2O → AOH + BH
Lipase,
amylase,
peptidase
Lyases
Non-hydrolytic addition or removal of
groups from substrates. C-C, C-N, CO or C-S bonds may be cleaved
RCOCOOH → RCOH + CO2
Decarboxylase
Isomerases
Intramolecule rearrangement, i.e.
isomerization changes within a single
molekule
AB → BA
Isomerase,
mutase
Ligases
Join together two molecules by
synthesis of new C-O, C-S, C-N or C-C
bonds with simultaneous breakdown
of ATP
X + Y+ ATP → XY + ADP + Pi
Synthetase
Example of EC numbering
1
1.1
1.1.1
1.1.1.1
1.1.1.2
Oxidoreductase
Acting on the CH-OH group of donors
With NAD or NADP as acceptor
NAD
NADP
Isozymes
• enzymes that differ in amino acid sequence but
catalyze the same chemical reaction
• usually display different kinetic parameters or
regulatory properties
• it permits the fine-tuning of metabolism to meet the
particular needs of a given tissue or developmental
stage
Cofactors - Coenzymes
Cofactors
Cofactors – other molecules bound to
enzymes to be fully active
• inorganic (Metal ions)
• organic compounds (Coenzymes)
• Coenzymes – may release from enzyme
and transfer from enzyme to enzyme
• Prosthetic groups - tightly bound to
enzymes
Examples of cofactors
Some inorganic ions that serve as
cofactors for enzymes
Coenzymes can serve as transient carriers of
specific atoms or functional groups
Coenzymes of oxidoreductases
Nicotinamide adenine dinucleotide (NAD+) - reversible
proton binding, catabolic (degradation) reactions
Coenzymes of oxidoreductases
Nicotinamide adenine dinucleotide phosphate (NADP+)
reversible proton binding,biosynthetic reactions
Coenzymes of oxidoreductases
Flavin adenine dinucleotide (FAD) is derived from riboflavin
(vitamin B2)
Coenzymes of oxidoreductases
Flavin mononucleotide (FMN), or riboflavin-5′phosphate, is a biomolecule produced from riboflavin
(vitamin B2)
Coenzymes of oxidoreductases
Iron-sulfur clusters are ensembles of iron and sulfide,
component of redox chains (respiratory, photosynthetic)
How enzymes work
Enzyme-Substrate Complex
Binding substrates in active sites
The surface of active site –
amino acid residues with
substituent groups binding
the substrate (together with
cofactors)
Transition State Theory
• Slow reactions face significant activation barriers
that must be surmounted during the reaction
Rate Acceleration
• The enzyme lowers the activation barrier compared
to the uncatalyzed aqueous reaction
How to Lower G?
Enzymes organizes reactive groups into
reaction favorable orientation
• Catalyzed reactions:
Enzyme uses the binding energy of substrates to organize the reactants
to a rigid ES complex
k1
k2
E + S  ES  E + P
k-1
k1[E][S] = (k-1+k2) [ES]
Two possible substrate enzyme
conformations
Lock and Key model
Induced fit model
How to Lower G?
Enzymes bind transition states best
– enzyme active sites are complimentary to the
transition state of the reaction
– enzymes bind transition states better than
substrates
– stronger interactions with the transition state as
compared to the ground state lower the
activation barrier
Support for TS Stabilization
Stable structural analogs of transition states bind more
strongly than reactants
Illustration of TS Stabilization Idea: Imaginary Stickase
Examples of enzymatic
reactions
Catalysis by fructose-2,6-bisphosphatase
(1) Lys 356 and Arg 257, 307, and
352 stabilize the quadruple negative
charge of the substrate by chargecharge interactions. Glu 327
stabilizes the positive charge on His
392.
(2) The nucleophile His 392 attacks
the C-2 phosphoryl group and
transfers it to His 258, forming a
phosphoryl-enzyme intermediate.
Fructose 6-phosphate leaves the
enzyme.
(3) Nucleophilic attack by a water
molecule, possibly assisted by Glu
327 acting as a base, forms
inorganic phosphate.
(4) Inorganic orthophosphate is
released from Arg 257 and Arg 307.
Enzyme kinetics
What is Enzyme Kinetics?
• Kinetics is the study of the rate at which
compounds react
• Rate of enzymatic reaction is affected by
– Enzyme
– Substrate
– Effectors
– Temperature, pH
How to Do Kinetic Measurements
Effect of Substrate Concentration
• The velocity of enzymatic reactions
depends on the substrate concentration
• Deviations due to:
– Limitation of measurements
– Substrate inhibition
– Contamination by inhibitors
Enzyme Kinetics Equations
Simplest Model Mechanism
One reactant, one product, no inhibitors
Simple reaction equation
In steady state - rate of ES production is
equal to its breakdown
Total enzyme concentration – the only we
know
Series of algebraic steps lead to an
expression of ES concentration
Concentration of ES depends on
[S], [ET] and a series of constants
Km – Michaelis constant
Concentration of ES depends on [S],
[ET] and Km – the Michaelis constant
Reaction rate depends on a concentration of
reactants ([ES] in our case) and a rate constant
In the case when
Reaction rate depend only on concentration of
substrate – first order reaction
In the case when
Most of the enzyme in ES state
Maximal reaction rate depends on total enzyme
concentration [ET]
• The final form in case of a single substrate is
k cat [ Etot ][ S ]
v
K m  [S ]
• kcat (turnover number): how many substrate
molecules can one enzyme molecule convert per
second
• Km (Michaelis constant): an approximate measure of
substrate’s affinity for enzyme
• Microscopic meaning of Km and kcat depends on the
details of the mechanism
Determination of Kinetic Parameters
Nonlinear Michaelis-Menten plot can be used to
calculate parameters Km and Vmax
Linearized double-reciprocal plot is good for
analysis of two-substrate data or inhibition
Enzyme Inhibition
Inhibitors are compounds that decrease enzyme’s activity
• Irreversible inhibitors (inactivators) react with the enzyme
- one inhibitor molecule can permanently shut off one enzyme molecule
- they are often powerful toxins but also may be used as drugs
• Reversible inhibitors bind to, and can dissociate from the enzyme
- they are often structural analogs of substrates or products
- they are often used as drugs to slow down a specific enzyme
• Reversible inhibitor can bind:
– To the free enzyme and prevent the binding of the substrate
– To the enzyme-substrate complex and prevent the reaction
Enzyme Inhibition
Three types of enzyme inhibition
• Competitive inhibition
• Uncompetitive inhibition
• Mixed inhibition
Competitive inhibitors bind to the enzyme's active site;
KI is the equilibrium constant for inhibitor binding to E
Uncompetitive inhibitors bind at a separate site,
but bind only to the ES complex;
KI′ is the equilibrium constant for inhibitor
binding to ES.
Mixed inhibitors bind at a separate site, but may bind to
either E or ES.
Introduction to metabolism
Life Needs Energy
• The ultimate source of this energy on the Earth is
the sunlight
At biochemical standard conditions (1M, pH 7, 298 K, 101.3
kPa) the free-energy change of a biochemical reaction is simply
an alternative expression of the equilibrium constant
Equilibrium constant measures the
direction of spontaneous processes
Actual free-energy changes depend
on reactant and product
concentrations
Standard equilibrium (K’eq) – initial concentrations of each
component is at 1M
This is not the case of living organism
Different concentrations of metabolites can affect the
reaction direction
In human erythrocytes
ATP = ADP + Pi
ATP = 2.25 mM
ADP = 0.25 mM
Pi = 1.65 mM
T = 37 oC (310 K)
G’o = - 30.5 kJ/mol
G = - 52 kJ/mol
Adenosin nucleotide and inorganic
phosphate concentrations in some cells
Complete Oxidation of Reduced
Compounds is Strongly Favorable
• This is how chemotrophs obtain most of their
energy
• In biochemistry the oxidation of reduced fuels
with O2 is stepwise and controlled
• Thermodynamically favorable is not the same
as being kinetically rapid – enzyme catalysis
Chemistry at Carbon
Covalent bonds can be broken in two ways
• Homolytic cleavage is rare
• Heterolytic cleavage is common but does not
occur for simple C-C bonds
Two mechanisms for cleavage of a C—C or C—H bond
radical formation
ion formation
Common chemical reactions chemical reactivity in biochemistry
Most reactions fall within few categories:
1. Cleavage and formation of C–C bonds
2. Internal rearrangements, isomerization, elimination
3. Group transfers (H+, CH3+, PO32-)
4. Oxidations-reductions (e- transfers)
1) Cleavage and formation of
C–C bonds
• Carbanions and carbcations – unstable
• In biochemical reactions - stabilisation of ionic
forms of carbon is essential
• Functional groups containing electronegative
atoms (O, N) alter the electronic structure of
adjacent carbon
• Stabilisation of carbionic intermediates
Chemical properties of carbonyl groups
• Introduction of carbonyl group
(or imine or special cofactors)
in particular location is
common in entire metabolism
2) Internal rearrangements,
isomerization, elimination
Redistribution of electrons that results in
alternations without a change in the overall
oxidation state of molecule
• Isomerization of fructose 6-phosphate from glucose 6phosphate
• Cis-trans isomerization in proteins, fatty acids
Isomerization of fructose 6-phosphare from
glucose 6-phosphate
3) Group Transfer Reactions
• Proton transfer - very common
• Methyl transfer - various biosyntheses
• Acyl transfer - biosynthesis of fatty acids
• Glycosyl transfer - attachment of sugars
• Phosphoryl transfer - to activate metabolites,
also important in signal transduction
Phosphoryl Transfer from ATP
• ATP is frequently the donor of the phosphate
in the biosynthesis of phosphate esters
4) Oxidation-Reduction Reactions
• Reduced organic compounds serve as fuels
from which electrons can be stripped off during
oxidation
• The flow of electrons can do work –
electomotive force (such as in electric motor,
bulb, etc.)
• During oxidation, electrons are transfered to
special electron carriers
The oxidation of carbon in biomolecules
o
x
i
d
a
t
i
o
n
Electron carriers
• A few types of coenzymes and proteins
serve as universal electron carriers
• Many biochemical oxidation-reduction
reactions involve transfer of two electrons
• In order to keep charges in balance, proton
transfer often accompanies electron
transfer
NAD and NADP as common
redox cofactors
• These are commonly called pyridine nucleotides
• They can dissociate from the enzyme after the
reaction
• In a typical biological oxidation reaction, hydride
(:H-) from an alcohol is transferred to NAD+ giving
NADH
• AH2 + NAD(P)+
A + NAD(P)H + H+
NAD and NADP in metabolism
NAD+/NADH - catabolism, further in ATP
production
NADP+/NADPH – anabolism, biosynthetic
reactions
Flavin Cofactors allow Single
Electron Transfers
• Flavoproteins (FMN, FAD)
• May participate in one- or two-electron
transfers
• Flavin cofactors are usually tightly bound to
proteins, some covalently
• Variability in reduction potentials
Special role of ATP in metabolism
Special role of ATP in metabolism
• stores energy obtained in catabolic
reactions
• transport the energy to compartments
or parts of organism where it is needed
• provides the energy for anabolic
biosynthetic processes
Chemical basis of large negative
free-energy of ATP
• Separation of negative charges on phosphate
oxygens upon ATP hydrolysis
• Resonance stabilization of phosphate products
• Ionisation of ADP product
• Better solvation of products
ATP provides energy by group transfer
Simple hydrolysis of ATP is not the
source of energy (only liberation of
heat)
• In most cases it is two-step
process:
1) Favorable ATP hydrolysis and Pi
transfer
2) Resonance stabilization of free Pi
• Some processes involve simple
hydrolysis:
- Binding ATP to a protein and its
hydrolysis – conformation change
of the protein – mechanical motion
Several Phosphorylated Compounds
Have Larger G’° Than ATP
• Again, electrostatic repulsion within the reactant,
molecule is relieved
• The products are stabilized via resonance, or by
more favorable solvation
• Possible tautomerization product
Hydrolysis of phosphoenolpyruvate (PEP)
Hydrolysis of 1,3 bisphosphoglycerate
Hydrolysis of phosphocreatine
Substrate level phosphorylation
Phosphorylated molecules with higher ΔG°’ can be
used to synthesize ATP
PEP + ADP = Pyruvate + ATP
ΔG°’ – 61,9 kJ/mol
Hydrolysis of Thioesters
• Acetyl-CoA
Hydrolysis of Thioesters
• Hydrolysis of thioesters, such as acetyl-CoA is
strongly favorable
• Acetyl-CoA is an important donor of acyl groups
– Feeding two-carbon units into metabolic pathways
– Synthesis of fatty acids
Hydrolysis of acetyl-coenzyme A
Molecular Basis of Thioester Reactivity
The orbital overlap between O and C atoms allows
resonance stabilization in oxygen esters