METABOLISMO DE PROTEINAS

Dr. Francisco J Valenzuela

 Fijación Nitrógeno
• Nitrogeno es un
elemento esencial de
las Biomoleculas
(aminoacidos,
nucleotidos,
proteinas y el DNA).

• La fijación del
nitrogeno (N2 a NH3),
es un proceso
reductivo realizado
por microorganismos
en relación simbiotica
con las plantas.
Fijación de nitrógeno
 Fijación del nitrogeno
• Se produce en bacterias como Klebsiella y Azotobacter, cyanobacteria y
Rhizobium.

• La reacción de reducción se puede resumir:

N2 + 8e- + 16ATP + 16H2O -> 2NH3 + H2 + 16ADP + 16 Pi + 8 H+

• The enzyme system for nitrogen fixation consists of two separate proteins.

• Both proteins contain iron-sulfur clusters. Nitrogenase also contains a

tightly bound iron-molybdenum cofactor. Some bacteria, such as
Azotobacter, contain more than one nitrogenase complex. Of the three
systems in Azotobacter, one uses vanadium instead of molybdenum and
another uses only iron.
Flujo en la fijación del nitrogeno.

Nitrogenase (also called component I or

molybdenum-iron protein) catalyzes the
reduction of N2

Nitrogenase reductase (also called

component II or iron protein),
transfers electrons from ferredoxin or
flavodoxin to nitrogenase
Structure of
nitrogenase
complex
Reactions in assimilation of ammonia and major
fates of the fixed nitrogen
 Utilization of Ammonia
• Ammonia is a central metabolite, but at high levels it is quite toxic. Ammonia is a substrate for five enzymes that convert it to various
organic nitrogen compounds. All organisms assimilate ammonia via reactions leading to glutamate, glutamine, asparagine, and
carbamoyl phosphate. Most of the nitrogen that finds its way from ammonia to amino acids and other nitrogenous compounds does
so via glutamate and glutamine. Thus, the amino nitrogen of glutamate and the amide nitrogen of glutamine are extremely active in
biosynthesis.

• Glutamate formation - Glutamate dehydrogenase (catalyzes the reductive amination of –ketoglutarate):

-Ketoglutarate + NH3 + NAD(P)H + 2H+ <=> Glutamate + H2O + NAD(P)

• Bacteria growing with ammonia as their sole nitrogen source use this reaction as the primary route for nitrogen assimilation. In animal
cells, the reversible reaction can function in either direction. The enzyme is allosterically regulated. ATP or GTP inhibits its action.

• Glutamate synthase catalyzes a similar reaction:

-Ketoglutarate + Glutamine + NADPH + H+ -> 2 Glutamate + NADP+

Glutamate synthase probably plays a larger role in glutamate synthesis in most cells than glutamate dehydrogenase, due to the high KM of the glutamate
dehydrogenase for NH3.

• Glutamine formation - Glutamine synthetase catalyzes the following reaction:

• Glutamate + NH3 + ATP -> Glutamine + ADP + Pi
• The E. coli glutamine synthetase is a dodecamer, with 12 identical subunits and the complex has a molecular weight of about 600,000 Daltons. The
amide nitrogen of glutamate is used for the synthesis of several amino acids, purine and pyrimidine nucleotides, and amino sugars, so glutamine
synthetase plays a central role in nitrogen metabolism. In animals, the enzyme is a key participant in detoxifying ammonia, particularly in the brain,
and in ammonia excretion in the kidney. Accumulation of glutamate and glutamine depletes -ketoglutarate, which would interfere with the citric acid
cycle. As a result, glutamine synthetase tightly regulated. Mechanisms include the following:
o Asparagine formation - Asparagine synthetase catalyzes the conversion of
aspartate to asparagine :
Aspartate + ATP + NH3 (Gln) -> Asparagine + AMP + PPi + (Glu)
.
• Carbamoyl phosphate formation - Carbamoyl Phosphate Synthetase
catalyzes the formation of carbamoyl phosphate in the following two
reactions (glutamine is the preferred substrate):
NH3 + HCO3- + 2ATP -> Carbamoyl Phosphate + 2ADP + Pi
Glutamine + HCO3- + 2ATP + H2O -> Carbamoyl Phosphate + 2 ADP + Pi +
Glutamate
two forms of the enzyme, one in mitochondria and one in cytoplasm are
found (eukaryotes,). The enzyme is inhibited by UTP, consistent with the
involvement of carbamoyl phosphate in pyrimidine nucleotide synthesis.
Reactions in assimilation of ammonia and major
fates of the fixed nitrogen
I. Glutamate Dehydrogenase: Reductive Amination of α-
ketoglutarate

Regulación alosterica (-) de ATP y GTP

(Glutamato sintasa)
 II. Glutamine sintetasa, regulación de la
actividad enzimatica
• Cumulative feedback Inhibition - Eight specific feedback inhibitors, which are either
metabolic end products of glutamine (tryptophan, histidine, glucosamine-6-phosphate,
carbamoyl phosphate, CTP, or AMP) or indicators of the general status of amino acid
metabolism (alanine or glycine), can bind to any of the subunits of the enzyme and at least
partially inhibit it. The more inhibitors that bind, the greater the inhibition.

• Covalent modification (adenylylation) - A specific tyrosine residue in glutamine synthetase

can react with ATP to form a phosphate ester with AMP. Adenylylation renders the catalytic
site of the enzyme inactive. Both processes are catalyzed by the same enzyme-a complex of
adenylyl transferase (AT) and a regulatory protein, PII. The form of PII determines whether
the AT-PII complex catalyzes adenylylation or deadenylylation. If PII is uridylyated, the AT-PII
complex catalyzes deadenylylation. Deuridylylation of PII causes the AT-PII complex to
catalyze adenylylation. The enzyme uridylyl transferase catalyzes uridylylation of PII, whereas
deuridylylation is catalyzed by a different enzyme. Uridylyl transferase is allosterically
regulated, with ATP and -ketoglutarate activating it and glutamine inhibits it.
Regulation of the activity of E. coli glutamine
synthetase
 III. Asparagine Synthetase
Asparagine synthetase catalyzes conversion of aspartate to asparagine:

Aspartate + ATP + NH3 (Glutamine) -> Asparagine + AMP + PPi + (Glutamate)

The reaction provides one way for cells to handle toxic ammonia and it also serves as a mechanism for
making asparagine. Glutamine is a preferred substrate over ammonia.
Note that asparagine synthetase cleaves ATP to yield AMP + PPi, whereas glutamine synthetase (another
enzyme for handling ammonia) yields ADP + Pi.
 IV. Carbamoyl Phosphate Synthetase

• Carbamoyl phosphate synthetase is an enzyme of the urea cycle that

catalyzes the following reaction:

NH4+ + CO2 + 2 ATP <=> Carbamoyl Phosphate + 2 ADP + Pi

Pero el donante de nitrógeno puede venir de dos fuentes

 Metabolic Nitrogen Balance

Most organisms have little capacity to store nitrogen. Thus,

animals must continually replenish nitrogen supplies through the diet
to replace nitrogen lost through catabolism. When dietary protein is
insufficient, proteins manufactured for other purposes, such as most
muscle proteins, are broken down and not replaced.
A well-nourished adult is said to be in nitrogen equilibrium or
normal nitrogen balance if:

o Dietary intake of nitrogen = Nitrogen loss (through excretion and other

processes, such as perspiration)
o Positive nitrogen balance occurs when:
oDietary intake of nitrogen > Nitrogen loss
o Negative nitrogen balance occurs when:
oDietary intake of nitrogen < Nitrogen loss
Origen biosintético de los aminoácidos

Organisms vary widely in their ability to

synthesize amino acids. Dietary amino
acid requirements for organisms vary
from all to none. Mammals require about
half of the amino acids in their diet for
growth and maintenance of normal
nitrogen balance . Amino acids that must
be provided in the diet to meet an
animal's metabolic needs are called
“essential amino acids”. Those that
need not be provided because they can be
biosynthesized in adequate amounts are
called “nonessential amino acids”.
 Transamination in Amino Acid Metabolism

o Transamination is the process by which an amino group, usually from glutamate, is transferred to an -keto acid, with
formation of the corresponding amino acid plus -ketoglutarate. Thus, transamination provides a route for redistribution of
amino acid nitrogen. Transamination reactions are catalyzed by transaminases (aminotransferases).
o Aminotransferases utilize a coenzyme - pyridoxal phosphate - which is derived from vitamin B6. The functional part of
pyridoxal phosphate is an aldehyde functional group attached to a pyridine ring. Catalysis involves a Schiff base intermediate
.
o Transamination reactions have equilibrium constants close to one. Therefore, the direction of a transamination reaction
proceeds in large part as a function of the intracellular concentrations of the reactants. This means that transamination can
be used not only for amino acid synthesis, but also for degradation of amino acids that accumulate in excess of need.
o Most aminotransferases use glutamate/-ketoglutarate as one of the two amino/keto acid pairs involved. Aminotransferases
involving aspartate/oxaloacetate and alanine/pyruvate are also quite abundant. Two important enzymes in the clinical
diagnosis of human disease are serum glutamate-oxaloacetate transaminase (SGOT) and serum glutamate-pyruvate
transaminase (SGPT):

SGOT: Oxaloacetate + Glutamate <-> Aspartate + -Ketoglutarate

SGPT: Glutamate + Pyruvate -------> -Ketoglutarate + Alanine

Abundant in heart and liver, are released as part of the cell injury that occurs in myocardial infarction,
infectious hepatitis, or other damage to either organ. Assays of these enzyme activities in blood serum can be
used both in diagnosis and in monitoring the progress of a patient during treatment.
Ojo: marcadores de????? Utilizados en la clínica
 DEGRADACION
• In a typical day, a person who is in nitrogen balance will consume 100 grams of protein, break down 400 grams of bodily protein,
resynthesize 400 grams of protein, and excrete/catabolize 100 grams. Proteins in extracellular environments, such as digestive
enzymes, polypeptide hormones, and antibodies, turn over quite rapidly, but proteins with predominantly structural roles, such as
collagen of connective tissue, are much more stable.
• Intracellular Proteases - Proteolytic enzymes are found throught the cell. Several proteases are present in the eukaryotic cytosol-two
Ca2+ activated proteases called calpains, a large multisubunit neutral protease, and a still larger ATP-dependent protease called the
proteasome . Lysosomal proteases, called cathepsins, are designed to function in an acidic milieu. Lysosomes form by budding from the
Golgi complex and are bags of digestive enzymes containing proteases, nucleases, lipases, and carbohydrate cleaving enzymes.
Lysosomes are involved in secretion of digestive enzymes, digestion of organelles destined for destruction, digestion of foodparticles or
bacteria engulfed by phagocytosis, and intracellular release of enyzmes followed by autolysis-digestion and death of the cell.

Four structural features are currently thought to be determinants of turnover rate:

• Ubiquitination - Ubiquitin is a 76-amino acid residue heat-stable protein found in all eukaryotic cells. An ATP-dependent reaction with
proteins links ubiquitin's C-terminal glycine to lysine amino groups in the target protein . Proteins modified in this way are degraded
soon afterward.
• Oxidation of amino acid residues - Conditions that generate oxygen radicals cause many proteins to undergo mixed-function oxidation
of particular residues. Conditions require Fe2+ and hydroxyl radical, and the amino acids most susceptible to oxidation are lysine,
arginine, and proline. E. coli and rat liver each contain a protease that cleaves oxidized glutamine synthetase in vitro, but does not
attack the native enzyme.
• PEST sequences - Virtually all short-lived proteins (i.e., half-lives less than 2 hours) contain one or more regions rich in proline,
glutamate, serine, and threonine. These regions are called PEST sequences because the one-letter codes for these amino acids are
P,E,S, and T, respectively. Very few longer-lived proteins contain these sequences. Furthermore, insertion of these sequences into long-
lived proteins increases their metabolic lability.

• N-terminal amino acid residue - An N-terminal protein residue of Phe, Leu, Tyr, Trp, Lys, or Arg is correlated with short metabolic
lifetimes. Proteins with other termini are far longer-lived. Thus, the intracellular half-life of a particular protein depends on the identity
of its N-terminal amino acid residue.
Vidas medias de proteínas
Como se ve un Proteosoma
 Pasos en la
degradación con
ubiquitina
Amino Acid Degradation

• The first step in amino acid degradation is removal of the -amino group. This
modification, usually a transamination, can also be used to generate glutamate
from -ketoglutarate via the glutamate dehydrogenase reaction. The products of
these reactions include deamination of the amino acid to the corresponding keto
acid plus ammonia. L-amino acid oxidase also catalyzes a similar reaction. Kidney
and liver cells are also rich in a D-amino acid oxidase, which has an unknown
function, because D isomers of amino acids are rare except for in bacterial cell
walls.
• After the nitrogen group has been removed from the molecules, the carbon
backbone can be metabolized in a variety of ways. Amino acids whose skeletons
generate pyruvate or oxaloacetate are efficiently converted to carbohydrates via
gluconeogenesis. Amino acids leading to acetyl-CoA or acetoacetyl-CoA
contribute towards ketogenesis. The terms glucogenic and ketogenic are used to
classify amino acids as generators of carbohydrates (see here) or ketone bodies,
respectively.
Amino Acid Degradation

H202 es un material toxico porque

produce estrés oxidativo
Fates of the amino acid carbon skeletons.
 Urea Cycle
• Accumulation Ammonia s toxic s. Because terrestrial animals must conserve water, they convert
ammonia to a form that can be excreted without large water losses. Birds, terrestrial reptiles, and
insects convert most of their excess ammonia to uric acid, an oxidized purine. Most mammals
excrete the bulk of their nitrogen as urea.
• Urea is synthesized in mammals almost exclusively in the liver and then transported to the kidneys
for excretion. The last step in the cycle is the hydrolytic cleavage of arginine to yield ornithine and
one molecule of urea. Virtually all organisms use the reactions to synthesize arginine from
ornithine, but they lack the arginase enzyme needed to make the pathway cyclic. Only ureotelic
organisms-those that excrete urea-contain arginase, so only ureotelic organisms can carry out the
cyclic pathway.

CO2 + NH4+ + 3 ATP + Aspartate + 2H2O -> Urea + 2 ADP + 2 Pi +AMP + PPi + Fumarate

• Reactions of the urea cycle occur in both the mitochondria and cytosol of liver cells. Glutamate
dehydrogenase, the citric acid cycle enzymes, carbamoyl phosphate synthetase I, and ornithine
transcarbamoylase are localized in the mitochondrion, whereas the rest of the cycle occurs in the
cytosol.
The Krebs-Henseleit urea cycle.
Biosynthesis of ornithine from
glutamate.
Transport of ammonia
Involvement of pyridoxal phosphate in
transamination.
Metabolic relationships among amino
acids derived from citric acid cycle
intermediates
 Synthesis and Catabolism of Glutamate,
Aspartate, Alanine, Glutamine, and Asparagine
Transport of ammonia to the liver for
urea synthesis.
Biosynthesis of ornithine from
glutamate
 Biosynthesis of creatine
phosphate and nitric oxide
from arginine.
 Biosynthesis of creatine
phosphate and nitric oxide from
arginine.
Mathematical modeling of the nitric oxide/cGMP pathway in the va
smooth muscle cell
Jin Yang , John W. Clark , Robert M. Bryan , Claudia S. Robertson
American Journal of Physiology - Heart and Circulatory PhysiologyPu
August 2005Vol. 289no. H886-H897DOI: 10.1152/ajpheart.00216.20
Biosintesis de prolina y acido Y-
aminobutirico
 Metabolism of Sulfur-Containing
Amino Acids
• SO42- + ATP <=> Adenosine-5'-Phosphosulfate
• Adenosine-5'-Phosphosulfate + ATP <=> 3'-
Phosphoadenosine-5'-Phosphosulfate (PAPS)
Outline of pathways
for cysteine and
methionine synthesis
in plants and
bacteria.
Animales: Outline of methionine
metabolism
Animales: Cystathionine as an intermediate
in cysteine biosynthesis.
Catabolic pathways for sulfur-containing amino acids
Biosintesis histidina
 Tyrosine Biosynthesis:
The phenylalanine hydroxylase and dihydropteridine reductase
reactions.
Catabolism of tyrosine to fumarate and
acetoacetate.
Metabolic fates of tryptophan.
FIN