Protein & Amino Acid

Metabolism

BBM FK Untar
October 2010
 INTRODUCTION
Biokimia dan Biologi Molekuler (BBM)
(Biochemistry and Molecular Biology)

• Biochemistry is the science of the molecules and chemical reaction of life.

• Biochemistry can be defined as the science of the chemical basis of life

(Gk bios "life"). Chemistry of life or life at the molecular level

• The cell is the structural unit of living systems.

• Thus, biochemistry can also be described as the science of the chemical
constituents of living cells and of the reactions and processes they
undergo.

• By this definition, biochemistry encompasses large areas of cell biology,

molecular biology, and molecular genetics.

• The lingua franca of the life sciences.

 • The Aim of Biochemistry Is to Describe &
Explain, in Molecular Terms, All Chemical
Processes of Living Cells

The major objective of biochemistry is the complete understanding, at the molecular

level, of all of the chemical processes associated with living cells. To achieve this
objective, biochemists have sought to isolate the numerous molecules found in cells,
determine their structures, and analyze how they function:

The Principal Methods and Preparations Used in Biochemical Labs:

1. Methods for Separating and Purifying Biomolecules:

- Salt fractionation,
- Chromatography (7)
- Electrophoresis
- Ultracentrifuge
2. Methods for Determining Biomolecular Structures:

- Elemental analysis
- Use of acid or alkaline hydrolysis to degrade the biomolecule
- Use of a battery of enzymes of known specificity to degrade biomolecule
- Visible , UV, infrared (IR), and NMR spectroscopy
- Mass spectrometry (MS)
- Specific sequencing methods (eg, for proteins & nucleic acids: WB,NB,SB)
- X-ray crystallography

3. Preparations for Studying Biochemical Processes :

- Whole animal (transgenic animals and animals with gene knockouts)

- Isolated perfused organ
- Tissue slice
- Whole cells
- Homogenate
- Isolated cell organelles
- Subfractionation of organelles
- Purified metabolites and enzymes
- Isolated genes (PCR and site-directed mutagenesis )
• A Knowledge of Biochemistry Is Essential to All Life Sciences
- Genetic, Physiology, Immunology, Pharmacology and pharmacy,
- Toxicology, Pathology, Microbiology, Zoology, Botany and etc.

• These relationships are not surprising, because life as we know it

depends on biochemical reactions and processes. In fact, the old barriers among
the life sciences are breaking down, and biochemistry is increasingly becoming
their common language ( lingua franca )

• A Reciprocal Relationship Between Biochemistry & Medicine Has

Stimulated Mutual Advances

The relationship between medicine and biochemistry

• The relationship between medicine and biochemistry has important implications
for the former. As long as medical treatment is firmly grounded in the knowledge of
biochemistry and other basic sciences, the practice of medicine will have a rational
basis that can be adapted to accommodate new knowledge.

• This contrasts with unorthodox health cults and at least some "alternative
medicine" practices that are often founded on little more than myth and wishful
thinking and generally lack any intellectual basis.

• NORMAL BIOCHEMICAL PROCESSES ARE THE BASIS OF HEALTH

• Biochemical Research Has Impact on Nutrition & Preventive Medicine

• Most & Perhaps All Diseases Have a Biochemical Basis
Summary
PROTEIN & AMINO ACID METABOLISM

 Amino Acids Metabolism

• Amino Acids Biosynthesis
• Amino Acids Catabolism
• Specialized Products

 Protein Metabolism
• Protein catabolism
• Protein Biosynthesis (not yet)
Introduction: Why Proteins are very Important

• Life on Earth depends on the biochemistry of two classes of

macromolecule: nucleic acids and proteins. Nucleic acids carry the genetic
information which is passed from parents to progeny, and which provides
the blueprint for a particular living organism. That blueprint is essentially a
set of instructions for making proteins.

• This obviously means that proteins are the key molecules in the
processes of life, and it is now known that virtually all the activities which
sustain living organisms are carried out by proteins.

• Proteins are constructed on a simple pattern, but that pattern allows for
an almost endless diversity of structure and function.

• Proteins are the most abundant biological macromolecules, occurring in

all
cells and all parts of cells.
The following are a few examples of proteins and what they do:

• All proteins, whether from the most ancient lines of bacteria or from the most
complex forms of life, are constructed from the same set of 20 amino acids,
covalently linked in characteristic linear sequences.
AMINO ACID METABOLISM
The Nitrogen Cycle and Nitrogen Fixation
 Nitrogen Fixation
1. Nitrogen fixation bacteria
• Free Bacteria: Klebsiella, Rhodoseudomonas, Chostridium,
Azobacter
• Symbiotic bacteria: Rhizobium (leguminous plants)

2. Reaction
• N2 → 2 NH3
• N2 + 6 e- + 6 H+ → 2 NH3
• N2 + + 10 H+ + 8 e- + 16 ATP → 2 NH3 + 16 ADP + Pi + H2

3. Nitrogenase complex
• Nitrogenase component: molybdenum-iron protein (MoFe protein)
• Reductase component: iron protien ([4Fe-4S] cluster

4. Requirements
• Ferredoxin
• ATP
AMINO ACIDS
BIOSYNTHESIS OF AMINO ACIDS

• Plants and microorganisms can make all 20

amino acids and all other needed N metabolites
• In these organisms, glutamate is the source of N,
via transamination (aminotransferase) reactions
• Mammals can make only 10 of the 20 aas
• The others are classed as "essential" amino acids
and must be obtained in the diet
• All amino acids are grouped into families
according to the intermediates that they are made
from.
• Biosynthetic families of amino acids
In bacteria and plants
• Major metabolic precursors are shaded blue
• Amino acids that give rise to other amino
acids are shaded yellow.
• Essential amino acids are red underline
Synthesis of Amino Acids
The origins of the carbon skeletons of amino acids.

Biosynthesis of amino acids, showing the connections to

glycolysis/gluconeogenesis and the TCA cycle
Overview of the synthesis of the nonessential amino acids
• 10 amino acids may be produced from glucose through intermediates of
glycolysis or the TCA cycle.
• Tyrosine, is synthesized by hydroxylation of the essential AA phenylalanine.
BIOSYNTHESIS OF AMINO ACIDS
• Glutamate Dehydrogenase,
► Play Central Roles in Amino Acid
• Glutamine Synthetase,
Biosynthesis
• Aminotransferases

1. Ammonia Is Incorporated into Glutamate and Glutamine

2. Transamination Reactions
I. BIOSYNTHESIS OF NONESSENTIAL AMINO ACIDS
• 8 amino acids may be produced from intermediates of
glycolysis or the TCA cycle.
• Cys and Tyr : are formed from essential aminp acids.

1. Glutamate

Reductive amination of α-ketoglutarate is catalyzed by

glutamate dehydrogenase (GDH)
2. Glutamine

The amidation of glutamate to glutamine catalyzed by

glutamine synthetase
3 & 4. Alanine and Aspartate
4 & 5. Aspartate and Asparagine
6 ,7 & 8. Serine, Glycine and Cysteine
8. Cysteine

Biosynthesis of cysteine in mammals

Biosynthesis of cysteine from serine in many bacteria and plants

9 & 10. Proline and Arginine

Conversion of glutamate to proline and arginine.

11. Tyrosine

Two distinct enzymatic activities are involved.

Activity II catalyzes reduction of dihydrobiopterin by NADPH
Activity I the reduction of O2 to H2O and of Phe to Tyr
 II. BIOSYNTHESIS OF ESSENTIAL AMINO ACIDS
A. Aspartate Family
Lysine, Methionine, and Threonine
B. Branched chain
Valine, Leucine, and Isoleucine
C. Aromatic
Phenylalanine, Tyrosine, and Tryptophan

Synthesis of shikimate and chorismate

Biosynthesis of Phe and Tyr from chorismate in E. coli.
The biosynthesis of tryptophan from chorismate requires five enzymes. In the
first step, the amide nitrogen of glutamine is transferred to chorismate; elimination
of the hydroxyl group and the adjacent pyruvate moiety of chorismate produces
the aromatic compound anthranilate . Anthranilate accepts a phosphoribosyl
moiety from PRPP. Rearrangement of the ribose, decarboxylation, and ring closure
generate indole glycerol phosphate.

Anthranilate.

The final two reactions of tryptophan biosynthesis are catalyzed by tryptophan

synthase:
D. Histidine

Synthesis of histidine from phosphoribosyl pyrophosphate (PRPP) and ATP.

Histidine is derived from PRPP (5 C atoms), the purine ring of ATP (1 N and 1 C),
glutamine (1 N), and glutamate (1 N).
Protein Catabolism
• Proteins are continually synthesized and degraded in all cells, a process
called turnover.

• Turnover 1 – 2% of total body protein,

75 – 80% are reutilzed for new protein synthesis
20 – 25% of the nitrogen form s urea

• Their half-lives can vary from a few minutes to several weeks. but the half-life of
a given protein in different organs and species is generally similar.
Short HF (T1/2) have PEST sequences (ex: HMG CoA reductase ;t1/2= 0,5-2 h)

• Protein Degradation 2 ways:

1. Lysosomes (ATP independent ):
- Extra cellular & membrane-associated protein
- Long-lived IC proteins
2. Ubiquitin Proteasome System (UPS), ATP dependent
- Short-lived & abnormal protein
Ubiquitination and hydrolysis of a protein
Overview of amino acid catabolism in mammals. The amino groups and
the carbon skeleton take separate but interconnected pathways.
 Degradation of Amino Acids

In animals, amino acids undergo oxidative degradation

in three different metabolic circumstances:

1. During the normal synthesis and degradation of cellular proteins (protein turnover),
some amino acids that are released from protein breakdown and are not needed for
new protein synthesis undergo oxidative degradation.

2. When a diet is rich in protein and the ingested amino acids exceed the body’s needs
for protein synthesis, the surplus is catabolized; amino acids cannot be stored.

3. During starvation or in uncontrolled diabetes mellitus,when carbohydrates are either

unavailable or not properly utilized, cellular proteins are used as fuel.
Amino Acid Catabolism
• Amino acids obtained from the degradation of endogenous proteins or from the
diet can be used for the biosynthesis of new proteins.

• Amino acids not needed for the synthesis of proteins are catabolized in order to
make use of their nitrogen and their carbon skeletons.

• The first step in amino acid degradation is often removal of the group. Next, the
carbon chains are altered in specific ways for entry into the central pathways of
carbon metabolism.

• Removal of the group of an amino acid occurs in several ways. The amino acid
usually undergoes transamination with α--ketoglutarateto form an acid and
glutamate.

• The glutamate is oxidized to and ammonia by the action of mitochondrial

glutamate dehydrogenase.

• The net effect of these two reactions is the release of groups as ammonia and
the formation of NADH and α-keto acids:
Conversion of the carbon skeletons of amino acids to pyruvate, acetoacetate, acetyl
1. Six Amino Acids Are Degraded to Pyruvate

Amino acids catabolized to pyruvate are both ketogenic and glucogenic.

The six are: alanine serin
tryptophan glycine
cysteine threonine

2. Seven Amino Acids Are Degraded to Acetyl-CoA:

Trp, Phe, Tyr
Lys, Leu, Ile and Ala

3. Five Amino Acids Are Converted to α-Ketoglutarate

Glu, Gln, Arg
Pro, His

4. Four Amino Acids Are Converted to Succinyl-CoA

Met
Thr
Val
Ile
A. Alanine, Asparagine, Aspartate, Glutamate and Glutamine

• Ala, Asp and Glutamate are synthesized by reversible transamination Rx

Breakdown into: pyruvat, oxaloacetate and α-KG, respectively.

• Degradation of both glutamine and asparagine begins with their hydrolysis

to Glu and Asp, respectively. Catalyzed by specific catabolic enzymes :
glutaminase and asparaginase
B. Arginine, Histidine, and Proline
C. Glycine and Serine
D. Threonine
E. The Branched-Chain Amino Acids
F. Methionine
G. Cysteine

The major route of cysteine catabolism is a three-step pathway leading to pyruvate.

Therefore, cysteine is glucogenic. Cysteine is first oxidized to cysteinesulfinate, which
loses its amino group by transamination to form β-sulfinylpyruvat.
Nonenzymatic desulfurylation produces pyruvate
H. Phenylalanine, Tyrosine and Tryptophan
Tryptophan

Conversion of tryptophan to alanine and acetyl CoA.

I. Lysine
Summary of amino acid catabolism
Incidence of inherited diseases of amino acid metabolism.
[Note: Cystinuria is the most common genetic error of amino acid transport.]
Overview of catabolism of amino groups & Excretory forms of nitrogen
Urea cycle: The steps of the cycle are numbered 1 to 5
The Urea Cycle Converts Ammonia into Urea
• Discovered by Hans Krebs and Kurt Henseleit in 1932, (almost exclusively in liver).

• In the first reaction, carbamoyl phosphate reacts in the mitochondrion with

ornithine to form citrulline in a reaction catalyzed by ornithine transcarbamoylase.

• Citrulline is then transported out, in exchange for cytosolic ornithine.

• The second nitrogen atom destined for urea comes from aspartate and is
incorporated when citrulline condenses with aspartate to form argininosuccinate.

• Argininosuccinate is cleaved nonhydrolytically to form arginine plus fumarate

in an elimination reaction catalyzed by argininosuccinate lyase.

• Final reaction of the urea cycle, the guanidinium group of arginine is hydrolytically
cleaved to form ornithine and urea in a reaction catalyzed by arginase.
• The exchange of glucose and alanine between muscle and liver, called the
glucose–alanine cycle , provides an indirect means for muscle to eliminate nitrogen
and replenish its energy supply.

Glucose–alanine cycle
Amino Acids as Metabolic Precursors
A. Products Derived from Glutamate, Glutamine and Aspartate

• Glu and Gln, are important players in nitrogen assimilation

• Glu and Asp, are amino group donors in many transamination reactions
and required in the urea cycle.

• Gln and Asp are also required as precursors in both purine and pyrimidine
biosynthesis.

• Glu is required for synthesis of biologically active tetrahydrofolate.

B. Products Derived from Serine and Glycine
C. Synthesis of Nitric Oxide from Arginine

Conversion of arginine to nitric oxide and citrulline. NADPH is the source

of the five electrons.
 Summary
• Nitrogen is fixed in only a few species of bacteria and blue-green algae by the
nitrogenase-catalyzed reduction of atmospheric to ammonia. Plants and
microorganisms can reduce nitrate and nitrite to ammonia.

• Ammonia is assimilated into metabolites by the reductive amination of to glutamate,

catalyzed by glutamate dehydrogenase. Glutamine, a nitrogen donor in
many biosynthetic reactions, is formed from glutamate and ammonia by the action of
glutamine synthetase.

• The amino group of glutamate can be transferred to an acid in a reversible

transamination reaction to form and the corresponding acid.

• Pathways for the biosynthesis of the carbon skeletons of amino acids begin with simple
metabolic precursors such as pyruvate and citric acid cycle intermediates.

• Nonessential amino acids are those that animals can produce in quantities that are
sufficient for growth. These amino acids are generally formed by short, energetically
inexpensive pathways. Essential amino acids must be supplied
in the diets of animals; these amino acids are synthesized by bacteria and plants.
• In addition to their role in protein synthesis, amino acids serve as precursors in a
number of other metabolic pathways.

• Protein molecules in all living cells are continually synthesized and degraded.

• Amino acids obtained from protein degradation or directly from food can be
catabolized. Catabolism begins with deamination, followed by modification of the
remaining carbon chains for entry into the central pathways of carbon metabolism.

• The pathways for the degradation of amino acids lead to pyruvate, acetyl CoA, or
intermediates of the citric acid cycle. Amino acids that are degraded to citric acid
cycle intermediates are glucogenic. Those that form acetyl CoA are ketogenic.

• In mammals, most nitrogen is excreted as urea, which is formed by the urea cycle in
the liver. The carbon atom of urea is derived from bicarbonate. One amino group is
derived from ammonia, and the other from aspartate.

• The metabolism of glutamine in the kidneys produces the bicarbonate needed to

neutralize acids produced in the body.
. Thank you

Even George W. says thanks!

Interlocking regulatory
mechanisms in the biosynthesis
of several amino acids derived
from aspartate in E. coli.

This overall regulatory mechanism

is called sequential feedback inhibition