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LICENCIATURA EM BIOLOGIA
DISCIPLINA
BIOQUÍMICA
Ano Lectivo de 2013/2014
Aula nº 23
16 MAI 2014
Ricardo Boavida Ferreira
Lab 46
Metabolismo dos aminoácidos e proteínas. I. Catabolismo
Turnover e semi-vida de proteínas. Proteases. A via da ubiquitina-proteassoma e as vias
lisossomais/vacuolares de degradação de proteínas.
Catabolismo de aminoácidos. Transaminação, desaminação oxidativa e catabolismo do esqueleto
carbonado. Aminoácidos glucogénicos e cetogénicos. Ciclo da ureia. Animais amonotélicos,
uricotélicos e ureotélicos. Aminoácidos como precursores de outras biomoléculas.
Material de estudo: diapositivos das aulas, bibliografia recomendada e textos de apoio.
Metabolismo dos
aminoácidos e proteínas
I. Catabolismo
Protein Turnover and Amino Acid
Catabolism
Proteins are degraded into amino acids
Protein turnover is tightly regulated
First step in protein degradation is usually the
removal of the α-amino nitrogen
Ammonium ion is converted to urea in most
mammals
Carbon skeletons are converted into other major
metabolic intermediates
• Amino acids used for synthesizing proteins are
obtained by degrading other proteins.
– Proteins destined for degradation are labeled with
ubiquitin.
– Polyubiquitylated proteins are degraded by
proteasomes.
• Amino acids are also a source of nitrogen for
other biomolecules.
Unlike excess carbohydrates and lipids, which may
be stored in the human body as glycogen and/or
fat, excess amino acids cannot be stored.
Surplus amino acids are used for fuel.
– Amino acid carbon skeletons are converted to
Acetyl–CoA
Acetoacetyl–CoA
Pyruvate
Citric acid cycle intermediates
– The amino group nitrogen is converted to urea and
excreted.
Glucose, fatty acids and ketone bodies can be
formed from amino acids.
Catabolismo das proteínas
Dietary proteins are a vital source of amino
acids.
Discarded cellular proteins are another source
of amino acids.
Dietary Protein Degradation
• Dietary proteins are hydrolyzed to amino acids
and absorbed into the bloodstream.
Cellular Protein Degradation
• Cellular proteins are degraded at different
rates.
– Ornithine decarboxylase has a half-life of 11
minutes.
– Hemoglobin lasts as long as a red blood cell.
– γ-Crystallin (eye lens protein) lasts as long as the
organism does.
A degradação de proteínas:
Polipéptido +
(n-1)H2O

naminoácidos
ΔGO’ < 0
- Definição: é a hidrólise das proteínas nos seus aminoácidos constituintes.
É um processo exergónico.
- As três alterações do metabolismo proteico como resposta universal ao stresse
- O turnover de proteínas
- Definição
- O atraso do conhecimento sobre a degradação
de proteínas relativamente à síntese
Ks
- A semi-vida das proteínas
- Os modelos iniciais; a dupla marcação radioactiva
Proteínas
Aminoácidos
- Significado fisiológico do turnover de proteínas
- Sintese e degradação são processos endergónicos in vivo
KD
- Alteração rápida na concentração de proteínas
- Adaptação a novas condições ambientais
- Stresse
- Função em condições fisiológicas específicas, como crescimento, diferenciação, etc.
- Exemplos da degradação de proteínas numa folha senescente de trigo e de macieira;
- Exemplos da degradação de proteínas de reserva numa semente de trigo e na casca dos
ramos da macieira.
- Hidrólise de proteínas estruturalmente anómalas
- Porque é que as proteínas com erros na sua estrutura são perigosas para as células?
- Agregados proteicos em doenças neurodegenerativas
- Proteólise em agregados proteicos – coalhada do queijo, tofu, corpos de Lewy
- Utilização das proteínas e aminoácidos na gluconeogénese ou como substratos
respiratórios
Proteases:
Enzimas proteolíticas, proteases ou peptidases são enzimas que catalisam a reacção de
hidrólise de ligações peptídicas, normalmente ligações eupeptídicas.
No caso de ligações isopeptídicas, recebem a designação genérica de isopeptidases.
Peptide bond hydrolase
Enzima proteolítica, protease ou peptidase
Endo-acting peptide bond hydrolase
Endopeptidase = proteinase
Exo-acting peptide bond hydrolase
Exopeptidase
Proteases are the single class of enzymes which occupy a pivotal position with respect to
their applications in both physiological and commercial fields. Since proteases are
physiologically necessary for living organisms, they are ubiquitous, being found in a wide
diversity of sources such as plants, animals, and microorganisms.
From the physiological point of view, proteases are degradative enzymes which catalyze the
total hydrolysis of proteins. Alternatively, they may conduct highly specific and selective
modifications of proteins such as activation of zymogenic forms of enzymes by limited
proteolysis, blood clotting and lysis of fibrin clots, and processing and transport of secretory
proteins across the membranes.
From the commercial point of view, the 1998 estimated value of the worldwide sales of
industrial enzymes is $1 billion. Of the industrial enzymes, 75% are hydrolytic. Proteases
represent one of the three largest groups of industrial enzymes and account for about 60% of
the total worldwide sale of enzymes. Proteases have a long history of application in the food
and detergent industries, but exhibit a variety of other applications, such as, for example the
leather industry.
Protease nomenclature
According to the Nomenclature Committee of the International Union of Biochemistry and
Molecular Biology (IUBMB), proteases are classified in subgroup 4 of group 3 (hydrolases)
(E.C.3.4).
Proteases are classified on the basis of three major criteria:
(i) type of reaction catalyzed,
(ii) chemical nature of the catalytic site, and
(iii) evolutionary relationship with reference to structure
Proteases are grossly subdivided into two major groups, i.e., exopeptidases and
endopeptidases, depending on their site of action. Exopeptidases cleave the peptide bond
proximal to the amino or carboxy termini of the substrate, whereas endopeptidases cleave
peptide bonds distant from the termini of the substrate.
Based on the functional group present at the active site, proteases are further classified into
four prominent groups, i.e., serine proteases, aspartic proteases, cysteine proteases, and
metalloproteases. There are a few miscellaneous proteases which do not precisely fit into
the standard classification, e.g., ATP-dependent proteases which require ATP for activity and
the proteasomes (20S and 26S), protease complexes which exhibit three different peptidase
activities: trypsin-like, chymotrypsin-like and peptidylglutamyl-peptide hydrolysing activities.
Based on their amino acid sequences, proteases are classified into five different families and
further subdivided into “clans” to accommodate sets of peptidases that have diverged from a
common ancestor. Each family of peptidases has been assigned a code letter denoting the
type of catalysis, i.e., S, C, A, M, or U for serine, cysteine, aspartic, metallo-, or unknown type,
respectively.
Exopeptidases
The exopeptidases act only near the ends of polypeptide chains. Based on their site of action
at the N or C terminus, they are classified as amino- and carboxypeptidases, respectively.
Aminopeptidases act at a free N terminus of the polypeptide chain and liberate a single
amino acid residue, a dipeptide, or a tripeptide.
Carboxypeptidases act at C terminals of the polypeptide chain and liberate a single amino
acid or a dipeptide. Carboxypeptidases can be divided into three major groups, serine
carboxypeptidases, metallocarboxypeptidases, and cysteine carboxypeptidases, based on the
nature of the amino acid residues at the active site of the enzymes.
Endopeptidases are characterized by their preferential action at the peptide bonds in the
inner regions of the polypeptide chain away from the N and C termini. The presence of the
free amino or carboxyl group has a negative influence on enzyme activity.
The endopeptidases are divided into four subgroups based on their catalytic mechanism, (i)
serine proteases, (ii) aspartic proteases, (iii) cysteine proteases, and (iv) metalloproteases.
To facilitate quick and unambiguous reference to a particular family of peptidases, a code
letter denoting the catalytic type, i.e., S, C, A, M, or U (see above) followed by an arbitrarily
number has been assigned to each enzyme.
Serine proteases are characterized by the presence of a serine group in their active site. They
are numerous and widespread among viruses, bacteria, and eukaryotes, suggesting that they
are vital to the organisms. Serine proteases are found in the exopeptidase, endopeptidase,
oligopeptidase, and omega peptidase groups. Based on their structural similarities, serine
proteases have been grouped into 20 families, which have been further subdivided into about
six clans with common ancestors.
Serine proteases are recognized by their irreversible inhibition by 3,4-dichloroisocoumarin
(3,4-DCI), L-3-carboxytrans 2,3-epoxypropyl-leucylamido (4-guanidine) butane (E.64),
diisopropylfluorophosphate (DFP), phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine
chloromethyl ketone (TLCK). Some of the serine proteases are inhibited by thiol reagents
such as p-chloromercuribenzoate (PCMB) due to the presence of a cysteine residue near the
active site.
Aspartic acid proteases, commonly known as acidic proteases, are the endopeptidases that
depend on aspartic acid residues for their catalytic activity. Acidic proteases have been
grouped into three families.
The aspartic proteases are inhibited by pepstatin. They are also sensitive to diazoketone
compounds such as diazoacetyl-D,L-norleucine methyl ester (DAN) and 1,2-epoxy-3-(pnitrophenoxy)propane (EPNP) in the presence of copper ions
Cysteine/thiol proteases occur in both prokaryotes and eukaryotes. About 20 families of
cysteine proteases have been recognized. The activity of all cysteine proteases depends on a
catalytic dyad consisting of cysteine and histidine. Based on their side chain specificity, they
are broadly divided into four groups: (i) papain-like, (ii) trypsin-like with preference for
cleavage at the arginine residue, (iii) specific to glutamic acid, and (iv) others. Papain is the
best-known cysteine protease.
They are susceptible to sulfhydryl agents such as PCMB but are unaffected by DFP and
metal-chelating agents.
Metalloproteases are the most diverse of the catalytic types of proteases. They are
characterized by the requirement for a divalent metal ion for their activity. They include
enzymes from a variety of origins such as collagenases from higher organisms, hemorrhagic
toxins from snake venoms, and thermolysin from bacteria. About 30 families of
metalloproteases have been recognized, of which 17 contain only endopeptidases, 12 contain
only exopeptidases, and 1 (M3) contains both endo- and exopeptidases. Families of
metalloproteases have been grouped into different clans based on the nature of the amino
acid that complexes the metal-binding site.
Metalloproteases are inhibited by chelating agents such as EDTA but not by sulfhydryl agents
or DFP.
Well known examples of proteases:
Plant proteases
Papain is a traditional plant protease that is extracted from the latex of Carica papaya fruits,
which are grown in subtropical areas of west and central Africa and India.
Bromelain is prepared from the stem and juice of pineapples.
Keratinases. Some of botanical groups of plants produce proteases which degrade hair.
Digestion of hair and wool is important for the production of essential amino acids, such as
lysine, and for the prevention of clogging of wastewater systems.
Animal proteases
Trypsin (23.3 kDa) is the main intestinal digestive enzyme responsible for the hydrolysis of
food proteins. It is a serine protease and hydrolyzes peptide bonds in which the carboxyl
groups are contributed by the lysine and arginine residues. Protein trypsin inhibitors occur in a
variety of edible seeds, which must therefore be boiled before used for human consumption.
Based on the ability of protease inhibitors to inhibit the enzyme from the insect gut, this
enzyme has received attention as a target for biocontrol of insect pests.
Chymotrypsin (23.8 kDa) is found in animal pancreatic extract. It is specific for the hydrolysis
of peptide bonds in which the carboxyl groups are provided by one of the three aromatic
amino acids, i.e., phenylalanine, tyrosine, or tryptophan. It is used extensively in the
deallergenizing of milk protein hydrolysates.
Pepsin (34.5 kDa) is an acidic protease that is found in the stomachs of almost all vertebrates.
The active enzyme is released from its zymogen, i.e., pepsinogen, by autocatalysis in the
presence of hydrochloric acid. Pepsin is an aspartyl protease and resembles human
immunodeficiency virus type 1 (HIV-1) protease, responsible for the maturation of HIV-1. The
enzyme catalyzes the hydrolysis of peptide bonds between two hydrophobic amino acids.
Rennin or chymosin is a pepsin-like protease (EC 3.4.23.4) that is produced as an inactive
precursor, prorennin, in the stomachs of all nursing mammals. It is converted to active rennin
(30.7 kDa) by the action of pepsin or by its autocatalysis. It is used extensively in the dairy
industry to produce a stable curd with good flavor. The specialized nature of the enzyme is
due to its specificity in cleaving a single peptide bond in k-casein to generate insoluble para-kcasein and C-terminal glycopeptide.
Microbial proteases
Although proteases are widespread in nature, microbes serve as a preferred source of these
enzymes because of their rapid growth, the limited space required for their cultivation, and the
ease with which they can be genetically manipulated to generate new enzymes with altered
properties that are desirable for their various applications. Thus, microbial proteases account
for approximately 40% of the total worldwide enzyme sales.
Bacteria. Most commercial proteases, mainly neutral and alkaline, are produced by organisms
belonging to the genus Bacillus. Neutrase, a neutral protease, is insensitive to the natural
plant proteinase inhibitors and is therefore useful in the brewing industry. Bacterial alkaline
proteases are characterized by their high activity at alkaline pH, e.g., pH 10, and their broad
substrate specificity. Their optimal temperature is around 60 °C. These properties of bacterial
alkaline proteases make them suitable for use in the detergent industry.
Fungi. Fungal enzymes can be conveniently produced in a solid-state fermentation process.
They are particularly useful in the cheesemaking industry due to their narrow pH and
temperature specificities.
Virus. Viral proteases have gained importance due to their functional involvement in the
processing of proteins of viruses that cause certain fatal diseases such as AIDS and cancer.
They have been looked as targets to design potent inhibitors that can combat the relentlessly
spreading and devastating epidemic of AIDS.
Pepsin
Three-dimensional structure of
porcine pepsin. Pepsin was the
first globular protein crystal used
successfully in x-ray diffraction.
Three-dimensional
crystal structure of
human pepsin
complexed with
pepstatin.
Crystal
structure of
human pepsin
and its
complex with
pepstatin.
The two aspartyl
residues at the
active site of
pepsin.
HIV protease
Gold and red ribbon structure of HIV protease with Glaxo
Wellcome inhibitor in active site.
HIV protease with four drugs (from left
to right: Indinavir, Saquinavir, Ritonavir,
and Nelfinavir) in the enzyme’s active
site.
As vias de degradação de proteínas:
- A via da ubiquitina-proteassoma
- As vias lisossomais/vacuolares
Regulation of Protein Turnover
• The protein ubiquitin is
used to mark cellular
proteins for destruction.
Ubiquitin
• Ubiquitin is activated and attached to proteins by
the coordinated action of three enzyme families:
– E1 - Ubiquitin activating enzyme
– E2 - Ubiquitin-conjugating enyzme
– E3 - Ubiquitin-protein ligase
The human papilloma virus encodes for an E3 protein which targets
the p53 tumor suppressor protein in its host. 90% of the cervical
cancers are associtated with this type of activity.
Via da ubiquitina/proteassoma
Péptidos
Conjugação
(E3)
Substrato
Endocitose
Degradação lisossomal
Trânsito
Expressão/silenciação
de genes
Degradação
Monoubiquitilação
E3-ligase de ubiquitina-proteína
E1-enzima activador da Ub
Desubiquitilação
Desconhecido
Lys6
ADP
ATP do grupo
•Responsáveis pela selecçãotampa
daProteassoma
proteína
substrato;
(não-proteolítico)
•Activação
COOH da glicina
do
26S
•Cerca de 1300 genes na Arabidopsis;
terminal
C da Ub;
19S
base
•Pouco semelhantes entre
si, podem conter•Codificado
um de
+
por um único gene;Proteassoma 26 S
Ub-P
Lys29
vários motivos estruturais (HECT,
RING,aU-box,
SCF, VBC,de 2 isoformas.
Proteólise ?
•Existência
a
a
a
APC, BTB/POZ).
b
b
b
b
Ub
a
b
b
b
Lys48
a
a
base
Ligação da UbLys63
6
6
6
29
29
29
20S
b
Conjugação
Ub
Proteassoma 26 S
•Descoberta
em 1975,daproteína
abundante
de função ATP
Proteólise
a
desconhecida e fortemente conservada, existência ubíqua;
48
48
48
E3
19S
Tolerância a danos no DNA
ADP
Activação de cinases
Activação
Trânsito da Ub
Tradução
(não-proteolítico)
•Proteína de 76 resíduos de aa com 8565 Da
E2-enzima
conjugador
da Ub
tampa
63
63
63
na forma
precursores
•Formação de ésteres entre
E2 e Ub •Sintetizada
que vão fornecer
a Ubde
para
a ligação (poliUbs e proteínas de
E3
Proteína
fusão de Ub com proteínas
ribossomais)
isopeptídica;
Desubiquitilação
E3
Adaptado
de http://cellbio.med.harvard.edu/faculty/goldberg/
alvo
•Cerca
de 45 genes na Arabidopsis;
Desubiquitilação
Alfred Goldberg Homepage
•Grande semelhanças entre si com um domínio conservado UBC
1/2/2007
Adaptado de Voges, D., Zwickl, P. e Baumeister, W. (1999) Annu Rev
Biochem 68, 1015-68.
Polyubiquitin-tagged proteins are often targeted for proteasome-mediated degradation
Origin and function of ubiquitin-like proteins
Hochstrasser, M. (2009) Nature 458, 422-429. doi:10.1038/nature07958
The ubiquitin-proteasome pathway is responsible for the degradation of hundreds, and probably thousands, of
proteins. Many of these substrates are regulatory proteins, such as transcription factors or cell-cycle regulators;
others are misfolded or otherwise aberrant proteins that must be eliminated to prevent their aggregation or
toxicity. A polyubiquitin-modified protein is the form most commonly targeted to the proteasome. Ubiquitin
receptors either in the proteasome regulatory particle (RP, purple) of the 26S proteasome or adaptor proteins
that associate reversibly with both polyubiquitylated proteins and specific proteasomal subunits (not shown)
allow binding of the proteolytic substrate to the proteasome. As shown in the cut-away on the right, ATPases
within the RP unfold the substrate and translocate it into the 20S proteasome core particle (CP, blue and red
rings), which houses the proteolytic sites in an interior chamber. The substrate is cleaved to small peptides.
Ubiquitin itself is normally recycled by DUBs that bind to or are intrinsic to the RP.
Microfotografia de microscópio electrónico de transmissão do
proteassoma da planta aquática lentilha de água menor (Lemna minor)
É uma proteína com uma massa molecular de 700 kDa e um tamanho de cerca de 2 nm (500.000
vezes mais pequena que 1 mm). Em baixo está representado um esquema da sua estrutura.
The UPS and Parkinson’s disease
Parkinson’s disease
Parkinson's disease is characterized by the presence of Lewy bodies and the loss
of dopamine-producing neurons in substantia nigra that controls muscle movement.
The Lewy body is an abnormal protein-aggregate structure found in certain areas of
the brain. It contains a protein called α-synuclein, which plays the central role in
Parkinson's disease and other diseases involving Lewy bodies, such as dementia
with Lewy bodies, multiple system atrophy, and Hallervorden-Spatz disease.
α-Synuclein
α-Synuclein is a 140 amino acid residue protein abundantly expressed in
presynaptic terminals of vertebrates. One of its normal functions is to regulate
dopamine transporter activities. This protein contains an NAC region that is prone
to aggregate, especially under oxidative conditions. The aggregated α-synuclein
can inhibit the function of 26S proteasome, which is important for the clearance of
misfolded proteins and other target molecules. The dysfunction of proteasome will
contribute to cell death.
Two mutations, A53T and A30P, in α-synuclein have been identified in families with
early-onset familial Parkinson's disease. These mutations may accelerate the
aggregation of α-synuclein. It is also interesting to note that, even without mutation,
extra copies of the gene encoding α-synuclein can cause Parkinson's disease at an
average age of just 34.
Parkin
Parkin is the causative gene for an autosomal recessive form of Parkinson's
disease. The gene was discovered in 1998. The parkin gene contains 12 exons
spanning over 1.5 Mb and encodes a protein of 465 amino acid residues with a
molecular mass of approximately 52 kDa.
The parkin gene product, Parkin protein, is a a ubiquitin-protein ligase (E3), a
component of the UPS, for the clearance of misfolded proteins and other target
molecules. Mutations of the Parkin gene are associated with early onset
Parkinson's disease.
Lewy bodies
α-Synuclein, Parkin, synphilin-1 and ubiquitin represent the major components of
Lewy bodies.
Models to study Parkinson’s disease
Human neuroblastoma cells treated with rotenone.
Transformed Saccharomyces cereviseae (baker’s yeats) expressing α-synuclein.
Transgenic Drosophila melanogaster .
Catabolismo dos aminoácidos
We Are Here
Amino acid metabolism
Urea Cycle
Em geral, o catabolismo dos aminoácidos
inicia-se com a separação dos grupos amina
dos seus esqueletos carbonados.
A remoção dos grupos α-amina dos
aminoácidos para dar os 2-oxoácidos
correspondentes é conseguida à custa de
dois tipos de reacções:
- Transaminação;
- Desaminação oxidativa.
As reacções de transaminação são
catalisadas por enzimas genericamente
designadas
por
transaminases
ou
aminotransferases.
Participam na transferência de um grupo
amina de um aminoácido dador para um 2oxoácido receptor, com a formação de um
novo oxoácido e de um novo aminoácido.
A transaminação não resulta, por isso, na
remoção líquida de azoto dos aminoácidos.
Contudo, permite que os grupos amina dos
diversos aminoácidos se concentrem num só
aminoácido, o glutamato.
A reacção de desaminação redutiva é
catalisada
pela
enzima
glutamato
desidrogenase (GDH).
Esta reacção liberta o amónio do glutamato
e forma ácido 2-oxoglutárico.
35
Desaminação oxidativa:
Destino do grupo NH2
Vias de desaminação:
-transaminação (formação
de ácido L-glutâmico)
-desaminação oxidativa
ác. L-glutâmico
Destino do esqueleto
carbonado:
-Piruvato
-Acetil-CoA
-Acetoacetato
-α-Cetoglutarato
-Succinil-CoA
-Fumarato
-Oxaloacetato
Entram no metabolismo
central como intermediários
do ciclo do ácido cítrico
-fonte de energia
-fonte de carbono (síntese
de glucose, ácidos gordos,
cetonas)
Destino do NH4+
O NH4+ em excesso é
tóxico; é eliminado ou
utilizado na síntese
de outros
aminoácidos
Destino do grupo -NH2
Plantas
Animais
- Amoníaco: animais amonotélicos
- Ácido úrico: animais uricotélicos
- Ureia: animais ureotélicos
Disposal of Amino Acids Nitrogen:
Key Reactions
• Transamination reactions
• Oxidative deamination reactions
– Glutamate dehydrogenase
– Hydrolytic deamination
• Glutaminase
• Glutamine synthesis
Removal of Nitrogen
• The first step in amino acid degradation is often
the removal of the nitrogen.
– The liver is the major site of protein degradation in
mammals.
• Oxidative deamination produces α-keto acids,
which are degraded to other metabolic
intermediates.
Conversion to Ammonium Ions
• α–Amino groups are converted to ammonium
ions by the oxidative deamination of
glutamate
Transamination
• Generally these enzyme funnel amino groups
to α–ketoglutarate.
– Aspartate transaminase
– Alanine transaminase
Disposal of Amino Groups:
Transamination Reactions
• Often the first step of amino acid degradation
• Transfer of amino group from many amino acids to
limited number of keto acid acceptors
– Pyruvate <-> alanine
– Oxaloacetate <-> aspartate
– α-Ketoglutarate <-> glutamate
Disposal of Amino Groups:
Transamination Reactions
• Transamination reactions tend to channel amino
groups on to glutamate
– Glutamate’s central role in amino acid N metabolism
Oxidative Deamination
• Glutamate dehydrogenase
Disposal of Amino Groups:
Deamination Reactions
• Glutamate dehydrogenase
– oxidative deamination
– Important in liver where it releases ammonia for urea
synthesis
• Hydrolytic deamination
– Glutaminase & asparaginase
Oxidative Deamination
• In most terrestrial vertebrates the ammonium
ion is converted to urea.
Disposal of Amino Groups:
Glutamine Synthetase
• Important plasma transport form of nitrogen from
muscle
• Detoxification of ammonia
– Brain
– Liver
• Removes ammonia intestinal tract
– Bacterial deamination of amino acids
– Glutamine utilization in intestinal cells
Serine and Threonine
• The β–hydroxy amino acids,
serine and threonine, can be
directly deaminated:
Ammonium Ion
• Ammonium ion is converted into urea in most
terrestrial vertebrates (ureotelic animals):
Porquê todo este “problema” com o azoto resultante do catabolismo dos
aminoácidos?
- Porque os animais não o podem armazenar;
- Porque não se pode acumular, porque é tóxico.
O amónio
Porque é que o amónio é tóxico para as células?
Porque desacopla as cadeias de transporte de electrões, do mitocôndrio e do cloroplasto, isto é, permite a
continuação do fluxo de electrões sem a correspondente do ATP de acordo com a teoria quimiosmótica,
proposta em 1961, por Peter Mitchell.
No mitocôndrio: espaço perimitocondrial, [H+] alta, pH baixo; matriz mitocondrial, [H+] baixa, pH alto.
No cloroplasto: dentro dos tilacóides, [H+] alta, pH baixo; fora dos tilacóides (estroma do cloroplasto), [H+]
baixa, pH alto.
Teoria quimiosmótica: síntese de ATP pela passagem de H+ do lúmen dos tilacóides ou do espaço
perimitocondrial dos mitocôndrios, em resposta ao gradiente de pH.
Amónio (NH4+) e amoníaco (NH3): a membrana do tilacóide (como a membrana interna do mitocôndrio) é
impermeável ao H+, mas pewrmeável ao NH4+ e aso NH3.
Se [NH4+] no mitocôndrio (nas plantas, produzido pela fotorrespiração), citoplasma ou cloroplasto aumenta, a
forma NH3 predomina no estroma do cloroplasto devido ao pH alto. O NH3 entra no lúmen do tilacóide.
Devido ao pH ser aqui relativamente mais baixo, o NH3 tem tendência para captar um protão, transformandose em NH4+. Este sai do tilacóide e entra no estroma do cloroplasto, onde o pH é mais alto => o NH4+ perde o
protão e converte-se em NH3. Isto é, o NH3 entra no tilacóide e sai protonado => desfaz-se o gradiente
51
electroquímico de pH => não ocorre síntese de ATP.
O Basta, um herbicida potente, tem como substância activa a fosfinotricina, um análogo estrutural do ácidso Lglutâmico.
A fosfinotricina é um inibidor da enzima glutamina sintetase (GS), uma enzima que participa no ciclo da
glutamato sintase (ou ciclo GS/GOGAT) e que é responsábvel pela assimilação da grande quantidade de amónio
produzido na fotorrespiração. Ao inibir a enzima GS, o amónio acumula-se e mata as plantas pelo mecanismo
atrás indicado.
Há uma bactéria do solo, a Streptomyces hygroscopicus, que codificsa uma enzima, a fosfinotricina
acetiltransferase (PAT) que metaboliza prontamente a fosfinotricina. Um grupo de investigadores da empresa
Hoechst transferiram o gene que codifica a PAT desta bactéria para uma outra bactéria, a Agrobacterium
tumefaciens, que infecta plantas.
Durante a infecção, esta segunda bactéria injecta o seu DNA no hospedeiro, criando uma planta transgénica. Já
foi, assim, possível obter plantas modificadas geneticamente (ex. tabaco) que expressam o gene da PAT. Ao serem
pulverizadas com o herbicida Basta, estas plantas transgénicas conseguem metabolizar a fosfinotricina, não sendo
mortas, ao contrário de todas as outras ao seu redor.
52
Nos animais ureotélicos, o amónio libertado durante o catabolismo dos
aminoácidos é convertido em ureia no mitocôndrio e citoplasma das células do
fígado, pela acção sequencial de cinco enzimas
Eliminação do NH4+ pelo ciclo
da ureia, também conhecido por
ciclo da arginina-ureia, ciclo da
ornitina ou ciclo de KrebsHenseleit, em homenagem a Hans
Krebs e a Kurt Henseleit, que o
elucidaram em 1932:
ureia
Fumarato
arginase
Arginina-succinato liase
hidrólise
Arginina
Ornitina
Arginina-succinato
NH3
(organismos
aquáticos)
Amonotélicos
Ureia
(animais
vertebrados)
Ureotélicos
Acidic úrico
(aves, répteis
terrestres e
insectos)
Uricotélicos
Citrulina
Ornitina transcarbamoílase
condensação
Arginina-succinato sintetase
A arginina é o precursor imediato
da ureia
Carbamoil-P
Reacção Global:
CO2 + NH4+ + 3ATP + aspartato + 2H2O 
ureia + 2ADP + 2Pi + AMP + PPi + fumarato
aspartato
HCO3
-
Carbamoíl-1-fosfato
sintetase
The Urea Cycle: Reminder
Amino acid
metabolism
We Are Here
Detoxification of Ammonia by the
Liver: the Urea Cycle
• Amino acid N flowing to liver as:
– Alanine & glutamine
– Other amino acids
– Ammonia (from portal blood)
• Urea
– chief N-excretory compound
The Urea Cycle
Formation of Carbamoyl Phosphate
• Carbamoyl phosphate synthetase
– Free NH4 reacts with HCO3- to form carbamoyl
phosphate.
– Reaction is driven by the hydrolysis of two molecules
of ATP
Formation of Citrulline
• Ornithine transcarbamoylase
– Citrulline is formed from transfer of the carbamoyl
group to the γ-amino group of ornithine.
Formation of Argininosuccinate
• Condensation of citrulline with aspartate to
form argininosuccinate
– Two equivalents of ATP are required.
Formation of Arginine and Fumarate
• Argininosuccinase
– Cleaves argininosuccinate to form arginine and
fumarate
• Arginase
Formation of Urea
– The arginine is hydrolyzed to produce the urea
and to reform the ornithine.
– The ornithine reenters the mitochondrial matrix.
Urea Cycle Linked to
Citric Acid Cycle
• The urea cycle is linked to the
citric acid cycle: Kreb’s Bi-cycle!!
Detoxification of Ammonia by the Liver:
the Urea Cycle
– Contains all enzyme of
urea cycle
• Site of urea synthesis
• Kidney has all urea cycle
enzymes except arginase
– Site of arginine
synthesis
– Mitochondria
– CPS regulatory enzyme
Detoxification
of Ammonia by
the Liver: the
Urea Cycle
Flow of Nitrogen from Amino Acids
to Urea in Liver
Notas finais sobre o destino do amónio.
Só os organismos ureotélicos são capazes de catalisar a hidrólise da arginina,
catalisada pela arginase (reacção 5 do ciclo da ureia), a reacção responsável pela
natureza cíclica do ciclo da ureia.
A síntese da ureia é dispendiosa do ponto de vista energético, requerendo a
hidrólise de 4 moléculas de ATP por volta do ciclo – são necessárias duas
moléculas de ATP para converter o AMP formado na reacção 3 em ATP. Daqui
dizer-se que uma dieta excessivamente rica em proteína sobrecarrega o fígado.
O fumarato produzido é hidratado a malato e este oxidado a oxaloacetato pelas
enzimas do ciclo do ácido cítrico. O oxaloacetato é, depois, transaminado a
aspartato. Assim, ambos os átomos de azoto da ureia têm origem em
aminoácidos: um é derivado do amónio libertado por desaminação oxidativa
(reacção 1); o outro é fornecido pelo aspartato.
O bicarbonato fornece o átomo de carbono da ureia.
Embora a ureia represente o principal produto final do metabolismo do azoto nos
mamíferos terrestres, sabe-se que os ursos em hibernação podem utilizar a ureia
para a biossíntese de aminoácidos.
The 10-Minute Urea Cycle
“Backwards”
This tutorial will show you that the urea cycle, one
of the most important cycles in nitrogen metabolism, can
be mastered in 10 minutes. Follow the instructions and
convince yourself by the test at the end that you know this
cycle. Another important point of the tutorial is to show
you that learning structures are the key to developing a
confidence in biochemical pathways.
So, start your clock and click 1 for the first slide.
As with most biochemical pathways, the urea cycle should be
mastered by working backwards. Let’s start by drawing the last
component in the pathway …Arginine (click 1). As you study the structure
of arginine try to imagine all of the intermediates in the pathway built into
this one molecule. Perhaps some color will help. Arginine is the
immediate source of urea. Can you see urea in arginine. Oh, excuse
me!. Click 1 to see urea. Now, can you see urea in arginine (click 1). The
oxygen is obtained from H2O when the urea molecule is hydrolyzed free
by the enzyme arginase.
–
COO
O
C
H2N
+
H3N-C-H
NH2
CH2
CH2
Urea
CH2
+
Citrulline
NH
O
HN=C
NH2
Ornithine
After the urea is removed
by hydrolysis, what
remains is ornithine
(click 1). Ornithine
reacts with carbamoylPO4 to form citrulline
(click 1). Now that you
know how three
compounds fit into the
arginine molecule, its
time to assemble
arginine (click 1 to go
on).
Arginine assembly starts with ornithine (click 1). First, you must make
the carbamoyl-PO4 that condenses with ornithine (click 1). As you can see
below, carbamoyl-PO4 is assembled from NH4+ and CO2 using 2ATPs as an
energy source and the enzyme carbamoyl-phosphate synthetase I .
O
NH4+ + CO2 + 2ATP
+ 2ADP + Pi
C
H2N
OPO3=
COO-
Carbamoyl-PO4 reacts
with ornithine to form
citrulline (click 1).
Phosphate is liberated in the
reaction (click 1). Once
citrulline is formed , all you
have to do to make arginine
is to replace the oxygen on
the citrulline with a nitrogen
group (click 1).
Ornithine
COO-
+
H3N-C-H
+
H3N-C-H
CH2
CH2
CH2
CH2
CH2
NH
CH2
+ NH
3
O=C
NH2
Citrulline
+ Pi
In the second step of the pathway the oxygen on the citrulline is
replaced with a NH3 group from aspartate (click 1). A complex must be
formed that allows the transfer to occur smoothly (click 1). Forming the
complex requires ATP, but no phosphate group is transfered (click 1).
COO−
COO −
+
H3N-C-H
COO −
+
H3N-C-H
CH2
CH2
CH2
NH
+
H3N-C-H
+
ATP
AMP + PPi
CH2
CH2
COO −
CH2
COO
CH2
CH2
NH
H-C-N =C
O=C
NH2
COO
NH2
Now you can see arginine in the product. The molecue that forms is
called “argininosuccinate” Where’s the succinate? Click 1 to see.
We now made urea, but there is still a little work left. Cyclic
pathways, as their name implies, return back to the starting compounds.
When we split the succinate from the argininosuccinate, a pair of electrons
went with the NH3 group and we were left with “oxidized” succinate, better
known as “fumarate” (click 1). Sound familiar. It should. This is Krebs
cycle stuff and we are in the mitochondria. Our objective is turn fumarate
back into aspartate (click 1). To do this we must make OAA (click 1). To
make OAA we need L-malate (click 1). Bingo! Now all we need is to
coenzymes and cofactors to connect all of these intermediates as you can
see it happen.
COO–
HC
H2O
HO-C-H
COO–
COO–
COO–
NAD+ NADH
C=O
Glutamate a-Kg
+
H3N-C-H
CH
CH2
CH2
CH2
COO–
COO–
COO–
COO–
Fumarate
L-Malate
OAA
Aspartate
Check you watch. You just learned the urea cycle is less than 10 minutes.
Click 1 and see how well you can do on a short test.
See what you learned. Click for the answer after reading the question.
1. Name six a-amino acids that are required in the urea cycle.
There are three standard amino acids, arginine, aspartate and glutamate,
as well as three non-protein amino acids: ornithine, citrulline and
argininosuccinate. By standard is meant amino acids that appear in
proteins and are coded for in the genetic code.
2. What is the function of glutamate in the cycle?
Glutamate is needed to regenerate aspartate from oxaloacetate. It may
generally be regarded as the donor of both urea nitrogen atoms.
3. All told, how may ATPs are needed to make one molecule of urea?
Three* are needed. Two to make carbamoyl-phosphate and one to
provide energy for the aspartate condensation with citrulline.
4. Why is the urea cycle referred to as a “bicycle”?
There are actually two cycles going on. One takes ornithine to
arginine and returns arginine to ornithine. The second takes
fumarate from the argininosuccinate and returns it to aspartate.
*Na realidade são 4 moléculas de ATP por cada molécula de ureia sintetizada (ver slide nº 61).
Destino dos
esqueletos carbonados
Carbon Skeletons
• The carbon atoms of degraded amino acids
emerge as major metabolic intermediates.
– Degradation of the 20 amino acids funnel into 7
metabolic intermediates:
• Acetyl–CoA
• Acetoacetyl–CoA
• Pyruvate
• α-Ketoglutarate
• Succinyl–CoA
• Fumarate
• Oxaloacetate
Carbon Skeletons
Ketogenic
Glucogenic
Both
leucine
lysine
serine
threonine
aspartic acid
glutamic acid
asparagine
glutamine
glycine
alanine
valine
proline
histidine
arginine
methionine
cysteine
isoleucine
phenylalanine
tryptophan
tyrosine
Carbon Skeletons
Pyruvate Entry Point
Oxaloacetate Entry Point
• Aspartate
– Transamination to oxaloacetate
• Asparagine
– Hydrolysis to Aspartate + NH4+
– Transmination to oxaloacetate
α–Ketoglutarate Entry Point
• Five carbon amino acids
α–Ketoglutarate Entry Point
• Histidine
α–Ketoglutarate Entry Point
• Proline and Arginine
Succinyl–CoA Entry Point
• Methionine, Valine & Isoleucine
Succinyl–CoA Entry Point
• Methionine
– Forms S-Adenosylmethionine
Branched-chained Amino Acids
Aromatic Amino Acids
• Phenylalanine
Aromatic Amino Acids
• Tetrahydrobiopterin - electron carrier
Aromatic Amino Acids
• Phenylalanine & Tyrosine
Aromatic Amino Acids
• Tryptophan
Notas finais sobre o destino dos esqueletos carbonados.
Após remoção do azoto, o esqueleto carbonado dos aminoácidos é convertido em
sete metabolitos intermediários, os quais podem ser directamente oxidados a CO2 e
H2O no ciclo do ácido cítrico ou usados na síntese de glucose ou de ácidos gordos.
Aminoácidos glucogénicos são aqueles cujos esqueletos carbonados são convertidos
em piruvato ou em intermediários do ciclo do ácido cítrico e que podem, por isso, ser
utilizados na síntese de glucose pelas reacções da gluconeogénese.
Aminoácidos cetogénicos são aqueles cujos esqueletos carbonados são
metabolizados a acetil-CoA ou acetoacetato, precursores dos ácidos gordos e dos
corpos cetónicos.
Corpos cetónicos são compostos formados pela cetogénese no organismo, sendo o
acetoacetato e os produtos dele derivados, o ácido β-hidroxibutírico e a acetona
(CH3COCH3).
Com excepção das sementes oleaginosas em germinação e de alguns
microrganismos que possuem o ciclo do glioxilato, todos os outros organismos são
incapazes de sintetizar glucose a partir do acetil-CoA ou do acetoacetato.
Alguns aminoácidos, como a isoleucina, a fenilalanina, a tirosina e o triptofano são
simultaneamente glucogénicos e cetogénicos, uma vez que parte do seu esqueleto
carbonado é glucogénica e a outra parte é cetogénica.
Notar que alguns aminoácidos são glucogénicos numas condições e cetogénicos
noutras.
FIM
The 10-Minute Urea Cycle
This tutorial will show you that the urea cycle,
perhaps the most important cycle in nitrogen metabolism,
can be mastered in 10 minutes. Follow the instructions
and convince yourself by the test at the end that you
know this cycle. Another important point of the tutorial is
to show you that learning structures are the key to
developing a confidence in biochemical pathways.
So, start your clock and click 1 for the first slide.
As with most biochemical pathways, the urea cycle should be
mastered by working backwards. Let’s start by drawing the last
component in the pathway …Arginine (click 1). As you study the structure
of arginine try to imagine all of the intermediates in the pathway built into
this one molecule. Perhaps some color will help. Arginine is the
immediate source of urea. Can you see urea in arginine. Oh, excuse
me!. Click 1 to see urea. Now, can you see urea in arginine (click 1). The
oxygen is obtained from H2O when the urea molecule is hydrolyzed free
by the enzyme arginase.
–
COO
O
C
H2N
+
H3N-C-H
NH2
CH2
CH2
Urea
CH2
+
Citrulline
NH
O
HN=C
NH2
Ornithine
After the urea is removed
by hydrolysis, what
remains is ornithine
(click 1). Ornithine
reacts with carbamoylPO4 to form citrulline
(click 1). Now that you
know how three
compounds fit into the
arginine molecule, its
time to assemble
arginine (click 1 to go
on).
Arginine assembly starts with ornithine (click 1). First, you must make
the carbamoyl-PO4 that condenses with ornithine (click 1). As you can see
below, carbamoyl-PO4 is assembled from NH4+ and CO2 using 2ATPs as an
energy source and the enzyme carbamoyl-phosphate synthetase I .
O
NH4+ + CO2 + 2ATP
+ 2ADP + Pi
C
H2N
OPO3=
COO-
Carbamoyl-PO4 reacts
with ornithine to form
citrulline (click 1).
Phosphate is liberated in the
reaction (click 1). Once
citrulline is formed , all you
have to do to make arginine
is to replace the oxygen on
the citrulline with a nitrogen
group (click 1).
Ornithine
COO-
+
H3N-C-H
+
H3N-C-H
CH2
CH2
CH2
CH2
CH2
NH
CH2
+ NH
3
O=C
NH2
Citrulline
+ Pi
In the second step of the pathway the oxygen on the citrulline is
replaced with a NH3 group from aspartate (click 1). A complex must be
formed that allows the transfer to occur smoothly (click 1). Forming the
complex requires ATP, but no phosphate group is transfered (click 1).
COO−
COO −
+
H3N-C-H
COO −
+
H3N-C-H
CH2
CH2
CH2
NH
+
H3N-C-H
+
ATP
AMP + PPi
CH2
CH2
COO −
CH2
COO
CH2
CH2
NH
H-C-N =C
O=C
NH2
COO
NH2
Now you can see arginine in the product. The molecue that forms is
called “argininosuccinate” Where’s the succinate? Click 1 to see.
We now made urea, but there is still a little work left. Cyclic
pathways, as their name implies, return back to the starting compounds.
When we split the succinate from the argininosuccinate, a pair of electrons
went with the NH3 group and we were left with “oxidized” succinate, better
known as “fumarate” (click 1). Sound familiar. It should. This is Krebs
cycle stuff and we are in the mitochondria. Our objective is turn fumarate
back into aspartate (click 1). To do this we must make OAA (click 1). To
make OAA we need L-malate (click 1). Bingo! Now all we need is to
coenzymes and cofactors to connect all of these intermediates as you can
see it happen.
COO–
HC
H2O
HO-C-H
COO–
COO–
COO–
NAD+ NADH
C=O
Glutamate a-Kg
+
H3N-C-H
CH
CH2
CH2
CH2
COO–
COO–
COO–
COO–
Fumarate
L-Malate
OAA
Aspartate
Check you watch. You just learned the urea cycle is less than 10 minutes.
Click 1 and see how well you can do on a short test.
See what you learned. Click for the answer after reading the question.
1. Name five a-amino acids that are required in the urea cycle.
There are three standard amino acids, arginine, aspartate and glutamate,
as well as two rare amino acids: ornithine and citrulline. By standard is
meant amino acids that appear in proteins and are coded for in the
genetic code.
2. What is the function of glutamate in the cycle?
Glutamate is needed to regenerate aspartate from OAA.
3. All told, how may ATPs are needed to make one molecule of urea?
Three are needed . Two to make carbamoyl-phosphate and one to
provide energy for the aspartate condensation with citrulline.
4. Why is the urea cycle referred to as a “bicycle”?
There are actually two cycles going on. One takes ornithine to
arginine and returns arginine to ornithine. The second takes
fumarate from the argininosuccinate and returns it to aspartate.
Questões
1-
B
Hexocinase (EC 2.7.1.1)
C
D
D-Glucose
+ H3PO4  D-glucose-6-fosfato + H2O
ADP + H3PO4  ATP + H2O
ΔG0’ = +3,3 kcal/mol
ΔG0’ = +7,3 kcal/mol
1.1 – Identifique a reacção química e a via metabólica onde participam todos os elementos
representados de A a D.
1.2 – Classifique essa via no que respeita às seguintes características:
- Anabólica, catabólica ou anfibólica;
- Linear, cíclica, em espiral, ramificada divergente ou ramificada convergente.
1.3 – Quais os seus quatro principais produtos?
1.4 – Considerando a reacção química considerada em C, indique, justificando, qual dos compostos é mais
rico em energia; a glucose ou a glucose-6-fosfato?
1.5 – Com base em C e D, escreva a reacção catalisada pela hexocinase e calcule o respectivo valor de
energia livre padrão.
1.6 - Com base na reacção da alínea anterior, justifique o nome trivial de hexocinase atribuído a esta
enzima.
1.7 – Lembrando a equação:
ΔG0’ = - R T Ln Keq
Calcule a respectiva Keq, considerando uma temperatura de 25 ºC (R = 1,987 cal. mol-1.K-1).
Considere o catabolismo intracelular da hexocinase no corpo humano.
1.8 – Escreva a equação de degradação da hexocinase e indique o nome genérico das enzimas que a
catalisam. A hexocinase humana é composta por 917 resíduos de aminoácidos.
1.9 – Indique os dois passos principais, bem como o nome das enzimas envolvidas, que participam na
desaminação dos aminoácidos formados de acordo com a alínes anterior.
1.10 – Descreva sucintamente, justificando, os passos subsequentes do amónio libertado, até à sua
excreção do organismo
1.11 – Identifique os sete metabolitos em que são convertidos os esqueletos carbonados dos aminoácidos
proteicos resultantes do catabolismo da hexocinase e descreva resumidamente o seu percurso
catabólico.
2 - Faça uma legenda descritiva para cada uma das figuras, A e B. Justifique a necessidade do consumo de
ATP em B.