http://intranet.tdmu.edu.ua/data/kafedra/inte
rnal/chemistry/classes_stud/en/med/lik/ptn/2
/02.%20Basic%20principles%20of%20metabol
ism%20catabolism,%20anabolism..htm
2
Differences between prokaryotic and
eukaryotic cells
Feature Prokyryotic cells Eukaryotic cell
Organisms Bacterie, cyanobacteria,
unicellular
Protozoa, fungi, plants,
animals, multicellular
Cell size (uM) 1 – 10 µm 10 – 20 µm
Separated nucleus No Yes
Subcellular organels No Yes
Character of Chromosomes Circular DNA Linear DNA
Ribosome size 70S 80S
Presence of cytoskeleton No Yes
Cell division Lateral/binary fission Mitosis
DNA Free Connected with protiens
(histones)
Protein synthesis events In cytoplasm Cytoplasm, ER
Location of respiratory
enzymes
In plasmatic membrane In mitochondrial membrate
(inner)
Assign the following processes to the corresponding cell compartments:
Transport of ions and small molecules, steroid synthesis, detoxification reactions,
respiration, metabolism of glucose, protein biosynthesis, ATP synthesis, intracellular
digestions, fatty acids oxidation, DNA synthesis, receptors for small molecules of
hormones, modification and sorting of proteins, hydrogen peroxide degradation,
protein degradation, RNA synthesis and RNA procesing
Cell membrane: Transport of ions and small molecules, receptors for small
molecules of hormones
Cytoplasm: metabolism of glucose, protein biosynthesis, ATP synthesis,
Mitochondria: respiration, protein biosynthesis, ATP synthesis, fatty acids
oxidation, DNA synthesis
Nucleus: DNA synthesis, RNA synthesis, RNA processiong, protein
biosynthesis,
Rough ER: protein biosynthesis
Smooth ER: steroid synthesis, detoxification reactions,
Golgi apparatus: modification and sorting of proteins, glycosylation of proteins,
export of proteins
Lysozome: intracellular digestions
Proteasome: protein degradation
Peroxisome: hydrogen peroxide degradation
4
• The cell membrane (also known as the plasma membrane or
cytoplasmic membrane) is a biological membrane that separates the
interior of all cells from the outside environment.
• The cell membrane is selectively permeable to ions and organic
molecules and controls the movement of substances in and out of
cells.[3]
• The basic function of the cell membrane is to protect the cell from
its surroundings. It consists of the phospholipid bilayer with
embedded proteins (receptors, ion chanells, transporters).
• Cell membranes are involved in a variety of cellular processes such
as cell adhesion, ion conductivity and cell signalling and serve as the
attachment surface for several extracellular structures, including the
cell wall, glycocalyx, and intracellular cytoskeleton. Cell membranes
can be artificially reassembled
Cell membranes:
- plasmatic membrane
• Cations
• Anions
the equation of a reaction catalyzed by succinate
dehydrogenase (structures, names, cofactors)
• The Succinate Dehydrogenase 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.
the equation of a reaction catalyzed by lactate
dehydrogenase (structures, names, cofactors)
• LDH catalyzes the conversion of lactate to pyruvic acid and back, as it
converts NAD+ to NADH and back. A dehydrogenase is an enzyme that
transfers a hydride from one molecule to another.
• Lactate dehydrogenase catalyzes the interconversion of pyruvate and lactate
with concomitant interconversion of NADH and NAD+. It converts pyruvate, the
final product of glycolysis, to lactate when oxygen is absent or in short supply,
and it performs the reverse reaction during the Cori cycle in the liver. At high
concentrations of lactate, the enzyme exhibits feedback inhibition, and the rate
of conversion of pyruvate to lactate is decreased. It also catalyzes the
dehydrogenation of 2-Hydroxybutyrate, but it is a much poorer substrate than
lactate.
8
Mitochondria
• Mitochondria are semi-autonomous organelles that contain their own DNA, own
proteosynthetic apparatus and are wrapped in a double membrane.
• The outer membrane is relatively high penetrability, the inner membrane is almost
impermeable and therefore contains a number of protein carriers, which allow transfer of
the necessary substances.
• Besides the transporters are located inner mitochondrial membrane as well as enzymes of
the respiratory chain and ATP-synthase enzyme, which occurs aerobic ATP formation
phosphorylation.
•
The material filling the content of mitochondria is called the mitochondrial matrix. It takes
place in the series of important events, such as the Krebs cycle, urea synthesis, heme
synthesis, synthesis of ketones, β oxidation of fatty acids ...
9
Golgi apparatus
• Golgi apparatus consisting of tanks and
transport vesicles.
• This is a polarized organelle - we can
distinguish the trans-side (which are
received by agents - especially proteins for
editing and sorting) and cis-side on
which these substances are also
released.
• Part of the cellular endomembrane
system, the Golgi apparatus packages
proteins inside the cell before they are
sent to their destination; it is particularly
important in the processing of proteins
for secretion.
It is of particular importance in processing
proteins for secretion, containing a set of
glycosylation enzymes that attach various
sugar monomers to proteins as the proteins
move through the apparatus.
10
Lysosomes
• Lysosomes are organelles of the cell digestion.
• In its membrane comprises a hydrogen pump, which is involved in maintaining an
acidic pH within them.
• Distinguish between primary and secondary lysosomes.
• Primary are those that have not participated in the digestion process, and do not
contain remnants of organelles, proteins etc.., And their enzymes have not yet been
used.
• Secondary lysosomes are those that are already involved in digestion.
Most enzymes found in lysosomes, belongs to the group of hydrolases, and the
cleavage of various bonds on different molecules.
• They are structurally and chemically spherical vesicles containing hydrolitic
enzymes, which are capable of breaking down virtually all kinds of biomolecules,
including proteins, nucleic acids, carbohydrates, lipids, and cellular debris. They are
known to contain more than fifty different enzymes which are all active at an acidic
environment of about pH 5.
11
The enzyme - type bonds
• α-glucosidase cleaves α-glycosidic linkage between the glucosamine
• β-galactosidase cleaves the β-glycosidic linkage between galactose
• Hyaluronidase cleaves a bond between molecules of hyaluronic acid
• arylsulphatase cleaves the bond sulfoesterovou
• Lysozyme cleaves the glycosidic bond
• cathepsin cleave a peptide bond (the protease)
• collagenase cleaves the triple helix collagen chain
• elastase cleaves a peptide bond (the protease)
• ribonuclease cleaves ribonucleotides diester linkage between
• lipase cleaves the ester bond between glycerol and fatty acids
• phosphatase cleaves the ester linkage (cleaves phosphate)
• ceramidasa cleave the ester bond between the ceramide, and fatty acid
Peptide bond
• When two amino acids form a dipeptide through a peptide bond it is called
condensation. In condensation, two amino acids approach each other, with the
acid moiety of one coming near the amino moiety of the other. One loses a
hydrogen and oxygen from its carboxyl group (COOH) and the other loses a
hydrogen from its amino group (NH2). This reaction produces a molecule of
water (H2O) and two amino acids joined by a peptide bond (-CO-NH-). The two
joined amino acids are called a dipeptide.
• The peptide bond is synthesized when the carboxyl group of one amino acid
molecule reacts with the amino group of the other amino acid molecule,
causing the release of a molecule of water (H2O), hence the process is a
dehydration synthesis reaction (also known as a condensation reaction).
• The formation of the peptide bond consumes energy, which, in living systems, is
derived from ATP.[6] Polypeptides and proteins are chains of amino acids held
together by peptide bonds. Living organisms employ enzymes to produce
polypeptides, and ribosomes to produce proteins. Peptides are synthesized by
specific enzymes. For example, the tripeptide glutathione is synthesized in two
steps from free amino acids, by two enzymes: gamma-glutamylcysteine
synthetase and glutathione synthetase.[7][8]
13
Non covalent interactions
• Coherence of cells and their interaction with each other between molecules
(eg. interaction between molecules and receptor molecules and enzymes,
etc.)
• And similar interactions are based on non covalent interactions The most
important non covalent interactions are:
hydrogen bonds
electrostatic interactions
hydrophobic interactions
Their main use in various situations described by the following table:
Structure / system prevailing type of non-covalent interactions :
Proteins: Structure secondary hydrogen bonding
Proteins: the tertiary structure hydrophobic and electrostatic interactions
Proteins: Structure quaternary electrostatic interactions
DNA hydrogen bonds
Phospholipid bilayer hydrophobic interactions
Binding of the enzyme-substrate electrostatic interactions
Binding of antibody-antigen electrostatic interactions
Characterize the structure of heam and its binding to the globin chain.
A heme group consists of an iron (Fe) ion
(charged atom) held in a heterocyclic ring,
known as a porphyrin. This porphyrin ring
consists of four pyrrole molecules cyclically
linked together (by methine bridges) with
the iron ion bound in the center.[36] The iron
ion, which is the site of oxygen binding,
coordinates with the four nitrogen atoms in
the center of the ring, which all lie in one
plane. The iron is bound strongly
(covalently) to the globular protein via the N
atoms of the imidazole ring of F8 histidine
residue (also known as the proximal
histidine) below the porphyrin ring. A sixth
position can reversibly bind oxygen by a
coordinate covalent bond,[37] completing the
octahedral group of six ligands. Oxygen
binds in an "end-on bent" geometry where
one oxygen atom binds to Fe and the other
protrudes at an angle. When oxygen is not
bound, a very weakly bonded water
molecule fills the site, forming a distorted
octahedron.
saturation curve of hemoglobin and myoglobin, mark areas corresponding to
transport O2 in lungs and mixed venous blood in graph.
• Hemoglobin (tetramer, cooperation effect)
• Myoglobin (monomer)
Note how the hemoglobin dissociation curve is Sshaped,
or sigmoidal, in character. This abrupt
change in oxygen affinity over a small range of
oxygen concentration is almost like a switch, allowing
the hemoglobin to almost fully unload bound oxygen
at the tissues where it is needed. We shall see later
that a number of modifiers of hemoglobin function
act by altering the nature of this binding curve,
changing the affinity of the hemoglobin for oxygen.
By contrast to hemoglobin, which is found in
circulating red blood cells or erythrocytes, myoglobin
is found intracellularly in body tissues. To perform its
job, myoglobin (Mb) must effectively bind any
oxygen released from hemoglobin (Hb). Myoglobin
therefore needs to have a higher oxygen affinity than
hemoglobin. The oxygen binding curves for
myoglobin and hemoglobin are compared in the
figure at left.
The hemoglobin in the red blood cells and myoglobin inside tissues such as muscle cells have to act in tandem for
effective oxygen transport. We can examine these coordinated activities using the oxygen binding curves of the two
respiratory pigments. For hemoglobin the task is to become completely loaded with oxygen as the blood traverses
through the capillaries of the lungs. It must then be able to efficiently off-load oxygen when the blood flows past the
low oxygen environment of active tissues, such as heart muscles. The following graph illustrates the different oxygen
affinities of myoglobin and hemoglobin at different concentrations of oxygen (given as partial pressure of O2)
derivatives of hemoglobin
• hemoglobin (Hb),
• oxyhemoglobin (O2Hb),
• carboxyhemoglobin (COHb),
• methemoglobin (MetHb),
• and sulfhemoglobin (SHb)
Kinetic curve
Dependence of concetration of S
(substrate,) or P (product) on Time
Saturation curve
Dependence of speed of reaction (velocity) on
concetration of S (substrate)
Km
concetration of S (substrate)
–1/2 of max. velocity
Substrate specifity
• A hexokinase is an enzyme that phosphorylates hexoses (six-carbon sugars), forming hexose phosphate.
In most organisms, glucose is the most important substrate of hexokinases, and glucose-6-phosphate is
the most important product. Hexokinase can transfer an inorganic phosphate group from ATP to a
substrate.
• Hexokinases should not be confused with glucokinase, which is a specific isoform of hexokinase. While
other hexokinases are capable of phosphorylating several hexoses, glucokinase acts with a 50-fold lower
substrate affinity and its only hexose substrate is glucose.
• Glucokinase (EC 2.7.1.2) is an enzyme that facilitates phosphorylation of glucose to glucose-6-
phosphate.
• Glucokinase (GK) is a hexokinase isozyme, related homologously to at least three other hexokinases.[1]
All of the hexokinases can mediate phosphorylation of glucose to glucose-6-phosphate (G6P), which is
the first step of both glycogen synthesis and glycolysis. However, glucokinase is coded by a separate
gene and its distinctive kinetic properties allow it to serve a different set of functions. Glucokinase has a
lower affinity for glucose than the other hexokinases do, and its activity is localized to a few cell types,
leaving the other three hexokinases as more important preparers of glucose for glycolysis and glycogen
synthesis for most tissues and organs. Because of this reduced affinity, the activity of glucokinase, under
usual physiological conditions, varies substantially according to the concentration of glucose.[2]
Allosteric enzymes
Allosteric enzymes are enzymes that change their conformational
ensemble upon binding of an effector, which results in an apparent
change in binding affinity at a different ligand binding site. This "action at
a distance" through binding of one ligand affecting the binding of another
at a distinctly different site, is the essence of the allosteric concept.
Allostery plays a crucial role in many fundamental biological processes,
including but not limited to cell signaling and the regulation of
metabolism. Allosteric enzymes need not be oligomers as previously
thought,[1] and in fact many systems have demonstrated allostery within
single enzymes.[2] In biochemistry, allosteric regulation (or allosteric
control) is the regulation of a protein by binding an effector molecule at a
site other than the enzyme's active site.
The site to which the effector binds is termed the allosteric site. Allosteric
sites allow effectors to bind to the protein, often resulting in a
conformational change involving protein dynamics. Effectors that enhance
the protein's activity are referred to as allosteric activators, whereas those
that decrease the protein's activity are called allosteric inhibitors.
Allosteric enzymes
• multiple subunits often regulatory and catalytic
• the enzyme binds effector structurally distinct from the substrate often product
• binds to the allosteric sites - other than the active site
• binding causes a conformational change in the enzyme activity …. change - allosteric
activation or inhibition
Allosteric enzymes in methabolismus
• citrate synthase, isocitrate dehydrogenase, and αketoglutarate
dehydrogenase
• pyruvate dehydrogenase
• Phosphofructokinase (PFK is an inducible, highly regulated,
allosteric enzyme that is a key regulator of glycolysis. )
Biochemistry-4-3-enzymes-kinetics 22
Enzyme Inhibitor Types
• Inhibitors of enzymes are generally molecules which resemble or mimic a particular
enzymes substrate(s). Therefore, it is not surprising that many therapeutic drugs are
some type of enzyme inhibitor. The modes and types of inhibitors have been
classified by their kinetic activities and sites of actions.
• These include Reversible Competitive Inhibitors,
Reversible Non-Competitive Inhibitors, and Irreversible
Inhibitors
Biochemistry-4-3-enzymes-kinetics 23
• Competitive inhibitors compete with the substrate for
binding at the active site (as E + I). In the double
reciprocal plot for a competitive inhibitor acting at the
substrate site for the following reasons, notice with
increasing concentration of inhibitor, the Vmax does not
change; however, the Km of the substrate is
increased. This also reflects the reversible nature of
the inhibitor; there is always some concentration of
substrate which can displace the inhibitor.
Reversible Competitive Inhibition
Biochemistry-4-3-enzymes-kinetics 24
• Non-competitive inhibitors combine with both the enzyme (E +
I) and the enzyme-substrate (EI + S) complex. The inhibitor
binds to a site other that the substrate site, and is thus
independent of the presence or absence of substrate. This
action results in a conformational change in the protein that
affects a catalytic step and hence decreases or eliminates
enzyme activity (formation of P). Notice in the reciprocal plot, a
non-competitive inhibitor does not affect the binding of the
substrate (Km), but it does result in a decrease in Vmax. This
can be explained by the fact that since inhibitor bound to an
enzyme inactivates it, the more EI formed will lower [ES] and
thus lower the overall rate of the reaction Vmax.
Reversible Non-Competitive
Inhibition
Biochemistry-4-3-enzymes-kinetics 25
• Irreversible inhibitors generally result in the destruction or
modification of an essential amino acid required for enzyme
activity. Frequently, this is due to some type of covalent link
between enzyme and inhibitor. These types of inhibitors range
from fairly simple, broadly reacting chemical modifying reagents
(like iodoacetamide that reacts with cysteines) to complex
inhibitors that interact specifically and irreversibly with active site
amino acids. (termed suicide inhibitors). These inhibitors are
designed to mimic the natural substrate in recognition and
binding to an enzyme active site. Upon binding and some
catalytic modification, a highly reactive inhibitor product is
formed that binds irreversibly and inactivates the enzyme. Use
of suicide inhibitors have proven to be very clinically effective
Irreversible Inhibitors
Cofactor, describe function, vitamin form, type of reactions, where we can
find it in cell
Nicotinamide adenine dinucleotide (NAD) is a
cofactor found in all living cells. The compound is
a dinucleotide, because it consists of two
nucleotides joined through their phosphate
groups. One nucleotide contains an adenine base
and the other nicotinamide. Nicotinamide
adenine dinucleotide exists in two forms, an
oxidized and reduced form abbreviated as NAD+
and NADH respectively.
Nicotinamide adenine dinucleotide is involved in
redox reactions, carrying electrons from one
reaction to another. The coenzyme is, therefore,
found in two forms in cells: NAD+ is an oxidizing
agent – it accepts electrons from other molecules
and becomes reduced. This reaction forms
NADH, which can then be used as a reducing
agent to donate electrons. These electron
transfer reactions are the main function of NAD.
However, it is also used in other cellular
processes, the most notable one being a
substrate of enzymes that add or remove
chemical groups from proteins, in
posttranslational modifications.
In organisms, NAD can be synthesized from
simple building-blocks (de novo) from the
amino acids tryptophan or aspartic acid. In an
alternative fashion, more complex components
of the coenzymes are taken up from food as
the vitamin called niacin.
27
Cofactors - NAD+
NOVÁK, Jan. Biochemie I.
Brno: Muni, 2009, Enzymy s. 8
O
OH O
N
C ONH2
C H2 O P O P O
OH
O O
OH
C H2
H O
OH OH
N
N
N
N
NH2adenin
pyridinium
ribose
Diphosphor. acid
Adition of hydride anion
N-glycosidic bond
ester
anhydrid
ester
ribose
N-glycosidic bond
molecular chaperones are proteins that assist the covalent folding or unfolding and the
assembly or disassembly of other macromolecular structures. Chaperones are present when
the macromolecules perform their normal biological functions and have correctly
completed the processes of folding and/or assembly. The chaperones are concerned
primarily with protein folding. The first protein to be called a chaperone assists the
assembly of nucleosomes from folded histones and DNA and such assembly chaperones,
especially in the nucleus,[1][2] are concerned with the assembly of folded subunits into
oligomeric structures.[3]
chaperone
Mechanism of glucose transport into the cells - symport of glucose with Na+
The secondary active transport of glucose in the kidney is Na+ linked; therefore an Na+
gradient must be established. This is achieved through the action of the Na+/K+ pump,
the energy for which is provided through the hydrolysis of ATP. Three Na+ ions are
extruded from the cell in exchange for two K+ ions entering through the intramembrane
enzyme Na+/K+-ATPase; this leaves a relative deficiency of Na+ in the intracellular
compartment. Na+ ions diffuse down their concentration gradient into the columnar
epithelia, co-transporting glucose. Once inside the epithelial cells, glucose reenters the
bloodstream through facilitated diffusion through GLUT2 transporters.
2. Phosphoglucose Isomerase catalyzes:
glucose-6-P (aldose)  fructose-6-P (ketose)
The mechanism involves acid/base catalysis, with ring
opening, isomerization via an enediolate intermediate,
and then ring closure. A similar reaction catalyzed by
Triosephosphate Isomerase will be presented in detail.
H O
OH
H
OHH
OH
CH2OPO3
2
H
OH
H
1
6
5
4
3 2
CH2OPO3
2
OH
CH2OH
H
OH H
H HO
O
6
5
4 3
2
1
glucose-6-phosphate fructose-6-phosphate
Phosphoglucose Isomerase
3. Phosphofructokinase catalyzes:
fructose-6-P + ATP  fructose-1,6-bisP + ADP
This highly spontaneous reaction has a mechanism similar
to that of Hexokinase.
The Phosphofructokinase reaction is the rate-limiting step
of Glycolysis.
The enzyme is highly regulated, as will be discussed later.
CH2OPO3
2
OH
CH2OH
H
OH H
H HO
O
6
5
4 3
2
1 CH2OPO3
2
OH
CH2OPO3
2
H
OH H
H HO
O
6
5
4 3
2
1
ATP ADP
Mg2+
fructose-6-phosphate fructose-1,6-bisphosphate
Phosphofructokinase
metabolism of pyruvate, name of process, structure and name of substrate and
products, names of enzymes
34
Total energy yield of aerobic glycolysis
Reaction ATP yield
glucose 2 pyruvate (substrate level phosporylation)
2 NADH  2NAD+
2
4-6*
Aerobic glycolysis till pyruvate:
* Depending on shuttle type
(see lecture Respiratory chain)
Further conversions of pyruvate:
Reakce ATP yield
2 pyruvate  2 acetylCoA + 2 NADH
2 acetyl CoA  2 CO2 + 6 NADH + 2 FADH2
6*
2x 12
Total maximal energy yield 36-38 ATP
* (2x NADH to the resp. chain)
35
Energy yield of anaerobic glycolysis
Anaerobic glycolysis till pyruvate:
Reaction ATP yield
glucose 2 x pyruvate (substrate-level phosphorylation)
2 NADH  2NAD+
2
0
Formation and consumption of NADH at anaerobic glycolysis
Reaction Yield/loss NADH
2 glyceraldehyde-3-P  2 1,3-bisP-glycerát
2 pyruvate  2 lactate
In sum
+2
-2
0
36
•The energy yield of anaerobic glycolysis is only 2 ATP
from substrate level phosphorylation
• it is only small portion of the total energy conserved in molecule of glucose
• it has high significane at situations when
• supply of oxygen is limited
• tissue do not dispose of mitochondrias (ercs, leukocytes, ..)
• it is necessary to spare lactate for gluconeogenesis
is a metabolic pathway that results in the generation of glucose from certain noncarbohydrate
carbon substrates. From breakdown of proteins, these substrates include
glucogenic amino acids (although not ketogenic amino acids); from breakdown of lipids (such
as triglycerides), they include glycerol (although not fatty acids); and from other steps in
metabolism they include pyruvate and lactate.
Gluconeogenesis is one of several main mechanisms used by humans and many other animals
to maintain blood glucose levels, avoiding low levels (hypoglycemia). Other means include the
degradation of glycogen (glycogenolysis),[1] fatty acid breakdown.
gluconeogenesis takes place mainly in the liver and, to a lesser extent, in the cortex of the
kidneys. In ruminants, this tends to be a continuous process.[3] In many other animals, the
process occurs during periods of fasting, starvation, low-carbohydrate diets, or intense
exercise. The process is highly endergonic until it is coupled to the hydrolysis of ATP or GTP,
effectively making the process exergonic. For example, the pathway leading from pyruvate to
glucose-6-phosphate requires 4 molecules of ATP and 2 molecules of GTP to proceed
spontaneously. Gluconeogenesis is often associated with ketosis. Gluconeogenesis is also a
target of therapy for type 2 diabetes, such as the antidiabetic drug, metformin, which inhibits
glucose formation and stimulates glucose uptake by cells.[4] In ruminants, because
metabolizable dietary carbohydrates tend to be metabolized by rumen organisms,
gluconeogenesis occurs regardless of fasting, low-carbohydrate diets, exercise, etc.[5]
Gluconeogenesis (GNG)
Glycogen is a multibranched polysaccharide of glucose that serves as
a form of energy storage
In humans, glycogen is made and stored primarily in the cells of the liver and the muscles
hydrated with three or four parts of water.[3] Glycogen functions as the secondary longterm
energy storage,[citation needed] with the primary energy stores being fats held in adipose
tissue. Muscle glycogen is converted into glucose by muscle cells, and liver glycogen
converts to glucose for use throughout the body including the central nervous system.
Glycogen synthesis is, unlike its breakdown, endergonic - it requires the input of energy.
Energy for glycogen synthesis comes from uridine triphosphate (UTP), which reacts with
glucose-1-phosphate, forming UDP-glucose, in a reaction catalysed by UTP—glucose-1phosphate
uridylyltransferase. Glycogen is synthesized from monomers of UDP-glucose
initially by the protein glycogenin, which has two tyrosine anchors for the reducing end of
glycogen, since glycogenin is a homodimer. After about eight glucose molecules have been
added to a tyrosine residue, the enzyme glycogen synthase progressively lengthens the
glycogen chain using UDP-glucose, adding α(1→4)-bonded glucose. The glycogen branching
enzyme catalyzes the transfer of a terminal fragment of six or seven glucose residues from a
nonreducing end to the C-6 hydroxyl group of a glucose residue deeper into the interior of
the glycogen molecule. The branching enzyme can act upon only a branch having at least 11
residues, and the enzyme may transfer to the same glucose chain or adjacent glucose
chains.
intracellular degradation of proteins
Biochemistry-7-1-AA 40
General equation of transamination reaction
CH2CH2COOH
O
CHOOC+R CH
NH2
COOH
Amino acid 2-oxoglutarate
(common acceptor)
HOOC CH CH2CH2COOH
NH2
+R C
O
COOH
glutamate2-oxo acid
Aminotransferase (enzyme)
Pyridoxalphosphate (cofactor)
Biochemistry-7-1-AA 41
The transaminase enzymes• are important in the production of various amino acids, and measuring the
concentrations of various transaminases in the blood is important in the diagnosing and
tracking many diseases. Transaminases require the coenzyme pyridoxal-phosphate,
which is converted into pyridoxamine in the first phase of the reaction, when an amino
acid is converted into a keto acid. Enzyme-bound pyridoxamine in turn reacts with
pyruvate, oxaloacetate, or alpha-ketoglutarate, giving alanine, aspartic acid, or glutamic
acid, respectively. Many transamination reactions occur in tissues, catalysed by
transaminases specific for a particular amino/keto acid pair. The reactions are readily
reversible, the direction being determined by which of the reactants are in excess. The
specific enzymes are named from one of the reactant pairs, for example; the reaction
between glutamic acid and pyruvic acid to make alpha ketoglutaric acid and alanine is
called glutamic-pyruvic transaminase or GPT for short.
• Tissue transaminase activities can be investigated by incubating a homogenate with
various amino/keto acid pairs. Transamination is demonstrated if the corresponding new
amino acid and keto acid are formed, as revealed by paper chromatography. Reversibility
is demonstrated by using the complementary keto/amino acid pair as starting reactants.
After chromatogram has been taken out of the solvent the chromatogram is then treated
with ninhydrin to locate the spots.
• Two important transaminase enzymes are AST (SGOT) and ALT (SGPT), the presence of
elevated transaminases can be an indicator of liver damage. This discovery was made by
Fernando De Ritis, Mario Coltorti and Giuseppe Giusti in 1955 at the University of
Naples.[1][2][3]
Biochemistry-7-1-AA 42
NH3
CO2
2ATP
2ADP + P
O
C
O P
NH2
COOH
NH2 CH2
CH2
CH2
CH NH2
OH2
HOOC CH2 CH
NH2
COOH
O
NH2 C NH CH2
CH2
CH2
CH
COOH
NH2
NHCOOH
CH NH C NH CH2
CH2
CH2
CH NH2
COOH
CH2
COOH
HOOC
HC
CH
COOH
NH
NH2 C NH CH2
CH2
CH2
CH
COOH
NH2
OH2
O
C
NH2 NH2
fumarate
arginine
urea
ornithine
citrulline
aspartate
argininesuccinate
Carbamoyl phosphate
MATRIX
MITOCHONDRIA
CYTOPLAZM
Urea synthesis
The liver forms it by combining two
ammonia molecules (NH3) with a
carbon dioxide (CO2) molecule in the
urea cycle
Biochemistry-7-1-AA 44
Regeneration of Aspartate
• PRODUCTION transamination from oxalcetate
(enzyme AST – aspartateminotransferase
• OA- intermediate of CAC
• Substrate for TA
• Substrate for 2-oxoglutarate (one reaction of CAC)
OA Asp
Biochemistry-7-1-AA 45
Synthesa glutamine
glutaminsynthetase
2. way detoxication NH3
glutamine
COOH
CH
CH2
H2N
CH2
C
O NH2
COOH
CH
CH2
H2N
CH2
C
O OH
+ NH3
glutamate
ATP ADP + P
- H2O
Biochemistry-7-1-AA 47
GMD r is reversibe reaction
Dehydrogenation deamination glutamate
Hydrogenation amination 2-oxoglutarate
COOH
CHH2N
CH2
CH2
COOH
NAD
+
NADH H
+
+
COOH
CHN
CH2
CH2
COOH
COOH
CO
CH2
CH2
COOH
H2O
NH3
Main NH3 formation in tissue
3. Way of detoxication of NH3
glutamate
2-iminoglutarate
Biochemistry-8-2_lipids 48
48
-oxidation
•
1.dehydrogenation
(FAD)
• 2.hydratation
• 3
dehydrogenation
(NAD+)
• 4 transfer of acyl
to CoASH
Oxidation of FA
acyl-CoA dehydrogenase
2-enoyl-CoA hydratase
3-hydroxyacyl-CoA
dehydrogenase
thiolase
Biochemistry-8-2_lipids 49
Fatty acids are synthesized by a cyclic pathway whose reactions appear
very similar to a reversal of fatty acid oxidation reactions.
However, although the reactions appear to be similar, the pathways are
very different, using different enzymes, different cofactors, and different
regulatory controls.
Fatty acids are built from acetyl CoA molecules (two carbons at a time.) First,
acetyl CoA is activated by adding CO2 to form malonyl CoA:
Acetyl CoA Malonyl CoA
Fatty Acid Synthesis
+ ATP + HCO3
- (& biotin cofactor)
oxalacetate
citrate
Cis-aconitate
isocitrate
Respiratory chain
•Complex I (NADH coenzyme Q reductase; labeled I) accepts electrons from the Krebs
cycle electron carrier nicotinamide adenine dinucleotide (NADH), and passes them to
coenzyme Q (ubiquinone; labeled UQ), which also receives electrons from complex II
(succinate dehydrogenase; labeled II). UQ passes electrons to complex III (cytochrome
bc1 complex; labeled III), which passes them to cytochrome c (cyt c). Cyt c passes
electrons to Complex IV (cytochrome c oxidase; labeled IV), which uses the electrons
and hydrogen ions to reduce molecular oxygen to water.
Uncouplers and Inhibitors
Specific inhibitors were used to distinguish the electron transport system from the
phosphorylation system and helped to define the sequence of redox carriers along the
respiratory chain. If the chain is blocked then all the intermediates on the substrate side of
the block become more reduced, while all those on the oxygen side become more oxidized.
It is easy to see what has happened because the oxidized and reduced carriers often differ
in their spectral properties. If a variety of different inhibitors are available then many of the
respiratory carriers can be placed in the correct order.
12-regulation 56
oxidative decarboxylation of 2-oxo
glutharate (keto-glutharate),
5 cofactors
biochemistry-9-TCA 57
Isocitrate to -ketoglutarate: step 3
Enzyme: isocitrate dehydrogenase
1st NADH produced 1st CO2 removed
Oxidation of IC to aKG and CO2, Mn2+ in active site
biochemistry-9-TCA 58
Succinyl CoA formation: step4
Enzyme: -ketoglutarate dehydrogenase
2nd NADH produced 2nd CO2 removed
oxidation
-SH
biochemistry-9-TCA 59
Succinate formation: step5
Enzyme: succinyl CoA synthetase
GTP produced
GTP + ADP  GDP + ATP (NPTase)
12-regulation 60
The covalent modification of proteins
• Many proteins are modified by the covalent linking of groups that can affect their function
and/or localisation in the cell. Such covalent modifications occur after synthesis and
folding of the polypeptide component. The main types of covalent modification and their
functions are listed below.
• Methylation/acetylation of amino acids at the N-terminal tails of histone proteins in
eukaryotes can affect the structure of chromatin and ultimately gene expression.
Prokaryotes also use methylation as a means of directly regulating protein activities. For
example, the methylation of specific proteins controlling flagellar movement is an
important mechanism for the regulation of bacterial chemotaxis.
• Phosphorylation of proteins (catalysed by specific kinases) is a key regulatory mechanism
in eukaryotic intracellular signalling and in metabolic pathways.
• Lipidation of proteins (i.e. addition of lipid tails) targets them to cell membranes (plasma
membrane and cell organelles).
• Glycosylation is a feature of many extracellular proteins, whether secreted or on the cell
surface, and may offer the protein some protection against proteases.
• These modifications are catalysed by specific enzymes and can be reversed, permitting
regulation of the protein's function. This reversibility is particularly significant with respect
to protein phosphorylation
12-regulation 61
12-regulation 63
ketone bodies
No electrolyteanionanion
Na+
Cl-
HCO3
-
OA
K+
Ketone bodies are three water-soluble molecules that
are produced by the liver from fatty acids during
periods of low food intake (fasting) or carbohydrate
restriction for cells of the body to use as energy instead
of glucose. Two of the three are used as a source of
energy in the heart and brain while the third (acetone)
is a degradation breakdown product of acetoacetic
acid.
acetone,
acetoacetic
acid, and
beta-
hydroxybut
yric acid
H3C C CH3
O
H3C CH CH2 C
O
OH
OH
H3C C CH2
O
C
O
O H
- CO2
- 2H
+ 2H
-hydroxybutyric acid Acetoacetic acid aceton
12-regulation 64
Ketone bodies as source of energy
acetoacetate
acetoacetyl-CoA
succinyl-CoA: acetoacetate-CoA transferasa
H3C C CH2
O
COOH
sukcinyl-CoA sukcinát
H3C C CH2
O
C
SCoA
O
S
H
CoA
C
SCoA
O
H3C2
CC
Energy