Biochemistry - as science. Structure and
properties of enzymes. The mechanism of
enzymes activity. Isoenzymes. Classification
of enzymes. Basic principles of metabolism.
Common pathways of proteins,
carbohydrates and lipids transformation.
ENZYMES: CLASSIFICATION, STRUCTURE
Enzymes - catalysts of biological reactions
Accelerate reactions by a millions fold
Common features for enzymes and
inorganic catalysts:
1. Catalyze only thermodynamically possible
reactions
2. Are not used or changed during the reaction.
3. Don’t change the position of equilibrium and
direction of the reaction
4. Usually act by forming a transient complex with
the reactant, thus stabilizing the transition state
Specific features of enzymes:
1. Accelerate reactions in much
higher degree than inorganic
catalysts
2. Specificity of action
3. Sensitivity to temperature
4. Sensitivity to pH
Structure of enzymes
Enzymes
Complex or holoenzymes (protein part
and nonprotein part – cofactor)
Apoenzyme (protein
part)
Simple (only protein)
Cofactor
Prosthetic groups
Coenzyme
-usually small inorganic
molecule or atom;
-large organic
molecule
-usually tightly bound to
apoenzyme
-loosely bound to
apoenzyme
Example of prosthetic group
Metalloenzymes
contain firmly
bound metal ions
at the enzyme
active sites
(examples: iron,
zinc, copper,
cobalt).
Example of metalloenzyme: carbonic
anhydrase contains zinc
Coenzymes
• Coenzymes act as group-transfer reagents
• Hydrogen, electrons, or groups of atoms can be
transferred
Coenzyme classification
(1) Metabolite coenzymes - synthesized from
common metabolites
(2) Vitamin-derived coenzymes - derivatives of
vitamins
Vitamins cannot be synthesized by mammals, but
must be obtained as nutrients
Examples of metabolite coenzymes
ATP can donate
phosphoryl group
ATP
S-adenosylmethionine
donates methyl groups
in many biosynthesis
reactions
S-adenosylmethionine
5,6,7,8 - Tetrahydrobiopterin
Cofactor of nitric oxide synthase
Vitamin-Derived Coenzymes
• Vitamins are required for coenzyme synthesis
and must be obtained from nutrients
• Most vitamins must be enzymatically
transformed to the coenzyme
• Deficit of vitamin and as result correspondent
coenzyme results in the disease
NAD+ and NADP+
• Nicotinic acid (niacin) an nicotinamide are precursor of
NAD and NADP
• Lack of niacin causes the disease pellagra
NAD and
NADP are
coenzymes
for
dehydrogenases
FAD and FMN
• Flavin adenine dinucleotide (FAD) and Flavin
mononucleotide (FMN) are derived from riboflavin (Vit B2)
• Flavin coenzymes are involved in oxidation-reduction
reactions
FMN (black), FAD (black/blue)
Thiamine Pyrophosphate (TPP)
• TPP is a
derivative of
thiamine (Vit B1)
• TPP participates
in reactions of:
(1) Oxidative
decarboxylation
(2) Transketolase enzyme
reactions
Pyridoxal Phosphate (PLP)
• PLP is derived from Vit B6 family of vitamins
PLP is a coenzyme for enzymes catalyzing reactions involving amino
acid metabolism (isomerizations, decarboxylations, transamination)
Pyridoxal Phosphate (PLP)
• PLP is derived from Vit B6 family of vitamins
PLP is a coenzyme for enzymes catalyzing reactions involving amino
acid metabolism (isomerizations, decarboxylations, transamination)
Enzymes active sites
Substrate usually is relatively small
molecule
Enzyme is large protein molecule
Therefore substrate binds to specific
area on the enzyme
Active site – specific region in the
enzyme to which substrate molecule is
bound
Characteristics of active sites
 Specificity (absolute, relative (group),
stereospecificity)
 Small three dimensional region of the protein.
Substrate interacts with only three to five amino
acid residues. Residues can be far apart in sequence
 Binds substrates through multiple weak
interactions (noncovalent bonds)
 There are contact and catalytic regions in the
active site
Active site of lysozym consists of six amino acid
residues which are far apart in sequence
Active site contains functional groups (-OH, -NH, -COO etc)
Binds substrates through multiple weak interactions
(noncovalent bonds)
Theories of active site-substrate
interaction
Fischer theory (lock and key model)
The enzyme active site (lock) is able to accept only a
specific type of substrate (key)
Properties of Enzymes
Specificity of enzymes
1.Absolute – one enzyme acts only on one substrate
(example: urease decomposes only urea; arginase
splits only arginine)
2.Relative – one enzyme acts on different
substrates which have the same bond type
(example: pepsin splits different proteins)
3.Stereospecificity – some enzymes can catalyze
the transformation only substrates which are in
certain geometrical configuration, cis- or trans-
Sensitivity to pH
Each enzyme has maximum activity at a particular pH
(optimum pH)
For most enzymes the optimum pH is ~7 (there are
exceptions)
Sensitivity to temperature
Each enzyme has
maximum activity at a
particular
temperature (optimum
temperature)
-Enzyme will
denature above 4550oC
-Most enzymes have
temperature
optimum of 37o
Naming of Enzymes
Common names
are formed by adding the suffix –ase to the name
of substrate
Example:
- tyrosinase catalyzes oxidation of tyrosine;
- cellulase catalyzes the hydrolysis of cellulose
Common names don’t describe the chemistry of the
reaction
Trivial names
Example: pepsin, catalase, trypsin.
Don’t give information about the substrate,
product or chemistry of the reaction
Principle of the international classification
All enzymes are classified into six categories
according to the type of reaction they catalyze
Each enzyme has an official international name
ending in –ase
Each enzyme has classification number
consisting of four digits: EC: 2.3.4.2
First digit refers to a class of enzyme, second to a subclass, third – to a subsubclass, and
fourth means the ordinal number of enzyme in
subsubclass
The Six Classes of Enzymes
1. Oxidoreductases
• Catalyze oxidation-reduction reactions
- oxidases
- peroxidases
- dehydrogenases
2. Transferases
• Catalyze group transfer reactions
3. Hydrolases
• Catalyze hydrolysis reactions where water
is the acceptor of the transferred group
- esterases
- peptidases
- glycosidases
4. Lyases
• Catalyze lysis of a substrate, generating a
double bond in a nonhydrolytic, nonoxidative
elimination
5. Isomerases
• Catalyze isomerization reactions
6. Ligases (synthetases)
• Catalyze ligation, or joining of two substrates
• Require chemical energy (e.g. ATP)
Kinetic properties of enzymes
Study of the effect of substrate concentration on the rate of reaction
Rate of Catalysis
- At a fixed enzyme concentration [E],
the initial velocity Vo is almost linearly
proportional to substrate concentration
[S] when [S] is small but is nearly
independent of [S] when [S] is large
- Rate rises linearly as [S] increases and
then levels off at high [S] (saturated)
Leonor Michaelis and Maud Menten – first researchers
who explained the shape of the rate curve (1913)
During reaction enzyme molecules, E, and substrate
molecules, S, combine in a reversible step to form an
intermediate enzyme-substrate (ES) complex
E + S
k1
k-1
ES
k2
E + P
k-2
k1, k-1, k2, k-2 - rate constant - indicate the speed
or efficiency of a reaction
The Michaelis-Menten Equation
The basic equation derived by Michaelis and Menten to explain
enzyme-catalyzed reactions is
Vmax[S]
vo =
Km + [S]
Km - Michaelis constant;
Vo – initial velocity caused by substrate concentration,
[S];
Vmax – maximum velocity
Effect of enzyme concentration [E]
on velocity (v)
In fixed, saturating
[S], the higher the
concentration of
enzyme, the greater
the initial reaction
rate
This relationship will
hold as long as there
is enough substrate
present
Enzyme inhibition
In a tissue and cell different chemical agents
(metabolites, substrate analogs, toxins,
drugs, metal complexes etc) can inhibit the
enzyme activity
Inhibitor (I) binds to an enzyme and prevents
the formation of ES complex or breakdown it to
E+P
Reversible and irreversible
inhibitors
Reversible inhibitors – after combining with
enzyme (EI complex is formed) can rapidly
dissociate
Enzyme is inactive only when bound to inhibitor
EI complex is held together by weak,
noncovalent interaction
Three basic types of reversible inhibition:
Competitive, Uncompetitive, Noncompetitive
Reversible inhibition
Competitive inhibition
•Inhibitor has a structure similar to the substrate
thus can bind to the same active site
•The enzyme cannot differentiate between the
two compounds
•When inhibitor binds, prevents the substrate
from binding
•Inhibitor can be released by increasing substrate
concentration
Competitive inhibition
Example of
competitive
inhibition
Benzamidine
competes with
arginine for binding
to trypsin
Noncompetitive inhibition
• Binds to an enzyme site different from the active
site
• Inhibitor and substrate can bind enzyme at the same
time
•Cannot be overcome by increasing the substrate
concentration
Uncompetitive inhibition
• Uncompetitive inhibitors bind to ES not to free E
• This type of inhibition usually only occurs in
multisubstrate reactions
Irreversible Enzyme Inhibition
very slow dissociation of EI complex
Tightly bound through covalent or noncovalent
interactions
Irreversible inhibitors
•group-specific reagents
•substrate analogs
•suicide inhibitors
Group-specific reagents
–react with specific R groups of amino acids
Substrate analogs
–structurally similar to the substrate for the
enzyme
-covalently modify active site residues
Suicide inhibitors
•Inhibitor binds as a substrate and is initially
processed by the normal catalytic mechanism
•It then generates a chemically reactive
intermediate that inactivates the enzyme
through covalent modification
•Suicide because enzyme participates in its
own irreversible inhibition
Regulation of enzyme
activity
Methods of regulation of enzyme
activity
•
•
•
•
Allosteric control
Reversible covalent modification
Isozymes (isoenzymes)
Proteolytic activation
Allosteric enzymes
Allosteric enzymes have a second regulatory
site (allosteric site) distinct from the active
site
Allosteric enzymes contain more than one
polypeptide chain (have quaternary structure).
Allosteric modulators bind noncovalently to
allosteric site and regulate enzyme activity via
conformational changes
2 types of modulators (inhibitors or
activators)
• Negative modulator (inhibitor)
–binds to the allosteric site and inhibits the
action of the enzyme
–usually it is the end product of a biosynthetic
pathway - end-product (feedback) inhibition
• Positive modulator (activator)
–binds to the allosteric site and stimulates
activity
–usually it is the substrate of the reaction
Example of allosteric enzyme - phosphofructokinase-1
(PFK-1)
• PFK-1 catalyzes an early step in glycolysis
• Phosphoenol pyruvate (PEP), an
intermediate near the end of the pathway
is an allosteric inhibitor of PFK-1
PEP
Regulation of enzyme activity by
covalent modification
Covalent attachment of a molecule to an amino acid side
chain of a protein can modify activity of enzyme
Phosphorylation reaction
Dephosphorylation reaction
Usually phosphorylated enzymes are
active, but there are exceptions
(glycogen synthase)
Enzymes taking part in phosphorylation are called protein kinases
Enzymes taking part in
dephosphorylation are called
phosphatases
Isoenzymes (isozymes)
Some metabolic processes are regulated by enzymes that
exist in different molecular forms - isoenzymes
Isoenzymes - multiple forms of an enzyme which
differ in amino acid sequence but catalyze the same
reaction
Isoenzymes can differ in:
 kinetics,
 regulatory properties,
 the form of coenzyme they prefer and
 distribution in cell and tissues
Isoenzymes are coded by different genes
Example: lactate dehydrogenase (LDH)
Lactate + NAD+
pyruvate + NADH + H+
Lactate dehydrogenase – tetramer (four subunits)
composed of two types of polypeptide chains, M and H
There are 5 Isozymes of LDH:
 H4 – heart
 HM3
 H2M2
 H3M
 M4 – liver, muscle
• H4: highest affinity; best in aerobic environment
•M4: lowest affinity; best in anaerobic environment
Isoenzymes are important for diagnosis of different
diseases
Activation by proteolytic cleavage
• Many enzymes are synthesized as inactive precursors
(zymogens) that are activated by proteolytic cleavage
• Proteolytic activation only occurs once in the life of an
enzyme molecule
Examples of specific proteolysis
•Digestive enzymes
–Synthesized as zymogens in stomach and pancreas
•Blood clotting enzymes
–Cascade of proteolytic activations
•Protein hormones
–Proinsulin to insulin by removal of a peptide
Multienzyme Complexes and
Multifunctional Enzymes
• Multienzyme complexes - different enzymes
that catalyze sequential reactions in the same
pathway are bound together
• Multifunctional enzymes - different
activities may be found on a single,
multifunctional polypeptide chain
Metabolite channeling
• Metabolite channeling - “channeling” of
reactants between active sites
• Occurs when the product of one reaction is
transferred directly to the next active site
without entering the bulk solvent
• Can greatly increase rate of a reactions
• Channeling is possible in multienzyme complexes
and multifunctional enzymes
• Metabolism - the entire network of chemical
reactions carried out by living cells. Metabolism
also includes coordination, regulation and energy
requirement.
• Metabolites - small molecule intermediates in
the degradation and synthesis of polymers
Most organism use the same general pathway for extraction
and utilization of energy.
All living organisms are divided into two major classes:
Autotrophs – can use atmospheric carbon dioxide as a sole
source of carbon for the synthesis of macromolecules.
Autotrophs use the sun energy for biosynthetic purposes.
Heterotrophs – obtain energy by ingesting complex carboncontaining compounds.
Heterotrophs are divided into aerobs and anaerobs.
A sequence of reactions that has a specific purpose
(for instance: degradation of glucose, synthesis of
fatty acids) is called metabolic pathway.
Metabolic pathway may be:
(a) Linear
(b) Cyclic
(c) Spiral pathway
(fatty acid
biosynthesis)
Metabolic pathways can be grouped into two paths –
catabolism and anabolism
Catabolic reactions - degrade molecules to create
smaller molecules and energy
Anabolic reactions - synthesize molecules for cell
maintenance, growth and reproduction
Catabolism is characterized by oxidation reactions and
by release of free energy which is transformed to ATP.
Anabolism is characterized by reduction reactions and
by utilization of energy accumulated in ATP molecules.
Catabolism and anabolism are tightly linked together
by their coordinated energy requirements: catabolic
processes release the energy from food and collect it
in the ATP; anabolic processes use the free energy
stored in ATP to perform work.
Anabolism and catabolism are coupled by energy
Metabolic Pathways Are Regulated
• Metabolism is highly regulated to permit organisms
to respond to changing conditions
• Most pathways are irreversible
• Flux - flow of material through a metabolic pathway
which depends upon:
(1) Supply of substrates
(2) Removal of products
(3) Pathway enzyme activities
Feedback inhibition
• Product of a pathway controls the rate of its own
synthesis by inhibiting an early step (usually the first
“committed” step (unique to the pathway)
Feed-forward activation
• Metabolite early in the pathway activates an enzyme
further down the pathway
Stages of metabolism
Catabolism
Stage I. Breakdown of macromolecules (proteins,
carbohydrates and lipids to respective building
blocks.
Stage II. Amino acids, fatty acids and glucose
are oxidized to common metabolite (acetyl CoA)
Stage III. Acetyl CoA is oxidized in citric acid
cycle to CO2 and water. As result reduced
cofactor, NADH2 and FADH2, are formed which
give up their electrons. Electrons are transported
via the tissue respiration chain and released
energy is coupled directly to ATP synthesis.
Glycerol
Catabolism
Catabolism is characterized by convergence of three
major routs toward a final common pathway.
Different proteins, fats and carbohydrates enter the
same pathway – tricarboxylic acid cycle.
Anabolism can also be divided into stages, however the
anabolic pathways are characterized by divergence.
Monosaccharide synthesis begin with CO2,
oxaloacetate, pyruvate or lactate.
Amino acids are synthesized from acetyl CoA, pyruvate
or keto acids of Krebs cycle.
Fatty acids are constructed from acetyl CoA.
On the next stage monosaccharides, amino acids and
fatty acids are used for the synthesis of
polysaccharides, proteins and fats.
Compartmentation of Metabolic
Processes in Cell
• Compartmentation of metabolic processes
permits:
- separate pools of metabolites within a cell
- simultaneous operation of opposing metabolic
paths
- high local concentrations of metabolites
• Example: fatty acid synthesis enzymes (cytosol),
fatty acid breakdown enzymes
(mitochondria)
Compartmentation of metabolic processes
OXIDATIVE DECARBOXYLATION
OF PYRUVATE
Matrix of the mitochondria
contains pyruvate
dehydrogenase complex
OXIDATIVE DECARBOXYLATION OF PYRUVATE
Pyruvate formed in the aerobic conditions undergoes
conversion to acetyl CoA by pyruvate dehydrogenase
complex.
Pyruvate dehydrogenase complex is a bridge between
glycolysis and aerobic metabolism – citric acid cycle.
Pyruvate dehydrogenase complex and enzymes of
cytric acid cycle are located in the matrix of
mitochondria.
Entry of Pyruvate into the Mitochondrion
Pyruvate freely diffuses through the outer membrane of mitochondria through the channels formed by transmembrane proteins porins.
Pyruvate translocase, protein embedded into the inner
membrane, transports pyruvate from the intermembrane space
into the matrix in symport with H+ and exchange (antiport) for
OH-.
Conversion of Pyruvate to Acetyl CoA
• Pyruvate dehydrogenase complex (PDH complex) is
a multienzyme complex containing 3 enzymes, 5
coenzymes and other proteins.
Pyruvate
dehydrogenase
complex is giant,
with molecular
mass ranging
from 4 to 10
million daltons.
Electron micrograph of the
pyruvate dehydrogenase
complex from E. coli.
Enzymes:
E1 = pyruvate dehydrogenase
E2 = dihydrolipoyl acetyltransferase
E3 = dihydrolipoyl dehydrogenase
Coenzymes: TPP (thiamine pyrophosphate),
lipoamide, HS-CoA, FAD+, NAD+.
TPP is a prosthetic group of E1;
lipoamide is a prosthetic group of E2; and
FAD is a prosthetic group of E3.
The building block of
TPP is vitamin B1 (thiamin);
NAD – vitamin B5 (nicotinamide);
FAD – vitamin B2 (riboflavin),
HS-CoA – vitamin B3 (pantothenic acid),
lipoamide – lipoic acid
Pyruvate dehydrogenase complex is a classic example of
multienzyme complex
Overall reaction of pyruvate dehydrogenase complex
The oxidative decarboxylation of pyruvate catalized by
pyruvate dehydrogenase complex occurs in five steps.
Aerobic cells
use a
metabolic
wheel – the
citric acid
cycle – to
generate
energy by
acetyl CoA
oxidation
The Citric
Acid
Cycle
Synthesis of
glycogen
Glucose
Pentose phosphate
pathway
Glucose-6phosphate
Glycogen
Ribose, NADPH
Degradation of
glycogen
Gluconeogenesis
Glycolysis
Ethanol
Fatty Acids
The citric acid
cycle is the
final common
pathway for the
oxidation of fuel
molecules —
amino acids,
fatty acids, and
carbohydrates.
Pyruvate
Lactate
Acetyl Co A
Amino Acids
Most fuel
molecules
enter the
cycle as
acetyl
coenzyme A.
Names:
The Citric Acid
Cycle
Tricarboxylic
Acid Cycle
Krebs Cycle
In
eukaryotes
the reactions
of the citric
acid cycle
take place
inside
mitochondria
Hans Adolf Krebs.
Biochemist; born in Germany.
Worked in Britain. His
discovery in 1937 of the
‘Krebs cycle’ of chemical
reactions was critical to the
understanding of cell
metabolism and earned him
the 1953 Nobel Prize for
Physiology or Medicine.
An Overview of the Citric Acid Cycle
A four-carbon oxaloacetate condenses with a
two-carbon acetyl unit to yield a six-carbon
citrate.
An isomer of citrate is oxidatively
decarboxylated and five-carbon ketoglutarate is formed.
-ketoglutarate is oxidatively
decarboxylated to yield a four-carbon
succinate.
Oxaloacetate is then regenerated from
succinate.
Two carbon atoms (acetyl CoA) enter the
cycle and two carbon atoms leave the cycle
in the form of two molecules of carbon
dioxide.
Three hydride ions (six electrons) are
transferred to three molecules of NAD+, one The function of the citric acid
pair of hydrogen atoms (two electrons) is cycle is the harvesting of highenergy electrons from acetyl CoA.
transferred to one molecule of FAD.
1. Citrate Synthase
• Citrate formed from acetyl CoA and oxaloacetate
• Only cycle reaction with C-C bond formation
• Addition of C2 unit (acetyl) to the keto double bond
of C4 acid, oxaloacetate, to produce C6 compound,
citrate
citrate synthase
2. Aconitase
• Elimination of H2O from citrate to form C=C bond
of cis-aconitate
• Stereospecific addition of H2O to cis-aconitate to
form isocitrate
aconitase
aconitase
3. Isocitrate Dehydrogenase
• Oxidative decarboxylation of isocitrate to
a-ketoglutarate (a metabolically irreversible reaction)
• One of four oxidation-reduction reactions of the cycle
• Hydride ion from the C-2 of isocitrate is transferred to
NAD+ to form NADH
• Oxalosuccinate is decarboxylated to a-ketoglutarate
isocitrate dehydrogenase
isocitrate dehydrogenase
4. The -Ketoglutarate Dehydrogenase Complex
• Similar to pyruvate dehydrogenase complex
• Same coenzymes, identical mechanisms
E1 - a-ketoglutarate dehydrogenase (with TPP)
E2 – dihydrolipoyl succinyltransferase (with flexible
lipoamide prosthetic group)
E3 - dihydrolipoyl dehydrogenase (with FAD)
-ketoglutarate
dehydrogenase
5. Succinyl-CoA Synthetase
• Free energy in thioester bond of succinyl CoA is
conserved as GTP or ATP in higher animals (or ATP
in plants, some bacteria)
• Substrate level phosphorylation reaction
+
Succinyl-CoA
Synthetase
GTP + ADP
GDP + ATP
HS-
6. The Succinate Dehydrogenase Complex
• Complex of several polypeptides, an FAD prosthetic group and
iron-sulfur clusters
• Embedded in the inner mitochondrial membrane
• Electrons are transferred from succinate to FAD and then to
ubiquinone (Q) in electron transport chain
• Dehydrogenation is stereospecific; only the trans isomer is
formed
Succinate
Dehydrogenase
7. Fumarase
• Stereospecific trans addition of water to the
double bond of fumarate to form L-malate
• Only the L isomer of malate is formed
Fumarase
8. Malate Dehydrogenase
Malate is oxidized to form oxaloacetate.
Malate
Dehydrogenase