LICENCIATURA EM BIOLOGIA DISCIPLINA BIOQUMICA Ano Lectivo de

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–Co. A Acetoacetyl–Co. A 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)H 2 O 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 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 (20 S and 26 S), 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 (M 3) 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 k. Da) 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 k. Da) 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 k. Da) 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 k. Da) 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 p. H, e. g. , p. H 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 p. H 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: – E 1 - Ubiquitin activating enzyme – E 2 - Ubiquitin-conjugating enyzme – E 3 - Ubiquitin-protein ligase The human papilloma virus encodes for an E 3 protein which targets the p 53 tumor suppressor protein in its host. 90% of the cervical cancers are associtated with this type of activity.

Via da ubiquitina/proteassoma Péptidos Conjugação (E 3) Substrato Endocitose Degradação lisossomal Trânsito Expressão/silenciação de genes Degradação Monoubiquitilação E 3 -ligase de ubiquitina-proteína E 1 -enzima activador da Ub Desubiquitilação Desconhecido Lys 6 ADP ATP do grupo • Responsáveis pela selecçãotampa da. Proteassoma proteína substrato; (não-proteolítico) • Activação COOH da glicina do 26 S • Cerca de 1300 genes na Arabidopsis; terminal C da Ub; 19 S base • Pouco semelhantes entre si, podem conter • Codificado um de + por um único gene; Proteassoma 26 S Ub-Pmotivos estruturais (HECT, Lys 29 vários RING, U-box, SCF, VBC, Proteólise ? • Existência de 2 isoformas. APC, BTB/POZ). b b Ub base b b b Lys 48 Ligação da Ub. Lys 63 6 6 6 29 29 29 20 S b Proteassoma 26 S Conjugação Ub • Descoberta em 1975, daproteína abundante de função ATP Proteólise desconhecida e fortemente conservada, existência ubíqua; 48 48 48 E 3 Tolerância a danos no DNA ADP Activação de cinases Activação Trânsito da Ub Tradução (não-proteolítico) 19 S • Proteína de 76 resíduos de aa com 8565 Da E 2 -enzima conjugador da Ub tampa 63 63 63 Sintetizada na forma precursores • Formação de ésteres entre E 2 e Ub • que vão fornecer a Ubde para a ligação (poli. Ubs e proteínas de E 3 Proteína fusão de Ub com proteínas ribossomais) isopeptídica; Desubiquitilação E 3 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/nature 07958 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 26 S 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 20 S 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 k. Da 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 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 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 26 S 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, A 53 T and A 30 P, 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 cause Parkinson's disease at an average of just 34.

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 k. Da. The parkin gene product, Parkin protein, is a a ubiquitin-protein ligase (E 3), 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 2 oxoácido 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 NH 2 Vias de desaminação: -transaminação (formação de ácido L-glutâmico) -desaminação oxidativa ác. L-glutâmico Destino do esqueleto carbonado: -Piruvato -Acetil-Co. A -Acetoacetato -α-Cetoglutarato -Succinil-Co. A -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 NH 4+ O NH 4+ em excesso é tóxico; é eliminado ou utilizado na síntese de outros aminoácidos

Destino do grupo -NH 2 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, p. H baixo; matriz mitocondrial, [H+] baixa, p. H alto. No cloroplasto: dentro dos tilacóides, [H+] alta, p. H baixo; fora dos tilacóides (estroma do cloroplasto), [H+] baixa, p. H 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 p. H. Amónio (NH 4+) e amoníaco (NH 3): a membrana do tilacóide (como a membrana interna do mitocôndrio) é impermeável ao H+, mas pewrmeável ao NH 4+ e aso NH 3. Se [NH 4+] no mitocôndrio (nas plantas, produzido pela fotorrespiração), citoplasma ou cloroplasto aumenta, a forma NH 3 predomina no estroma do cloroplasto devido ao p. H alto. O NH 3 entra no lúmen do tilacóide. Devido ao p. H ser aqui relativamente mais baixo, o NH 3 tem tendência para captar um protão, transformandose em NH 4+. Este sai do tilacóide e entra no estroma do cloroplasto, onde o p. H é mais alto => o NH 4+ perde o protão e converte-se em NH 3. Isto é, o NH 3 entra no tilacóide e sai protonado => desfaz-se o gradiente 51 electroquímico de p. H => 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 NH 4+ pelo ciclo da ureia, também conhecido por ciclo da arginina-ureia, ciclo da ornitina ou ciclo de Krebs. Henseleit, em homenagem a Hans Krebs e a Kurt Henseleit, que o elucidaram em 1932: ureia Fumarato Arginina-succinato liase arginas ehidrólise Arginina Ornitina Arginina-succinato NH 3 (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: CO 2 + NH 4+ + 3 ATP + aspartato + 2 H 2 O ureia + 2 ADP + 2 Pi + AMP + PPi + fumarato aspartato HCO 3 - 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 NH 4 reacts with HCO 3 - 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 H 2 O when the urea molecule is hydrolyzed free by the enzyme arginase. O + C H 2 N NH 2 – COO H 3 N-C-H CH 2 Urea CH 2 Citrulline + O HN=C NH NH 2 Ornithine After the urea is removed by hydrolysis, what remains is ornithine (click 1). Ornithine reacts with carbamoyl-PO 4 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-PO 4 that condenses with ornithine (click 1). As you can see below, carbamoyl-PO 4 is assembled from NH 4+ and CO 2 using 2 ATPs as an energy source and the enzyme carbamoyl-phosphate synthetase I. O NH 4+ + CO 2 + 2 ATP + 2 ADP + Pi C H 2 N OPO 3= COO- Carbamoyl-PO 4 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- + H 3 N-C-H CH 2 CH 2 NH CH 2 + NH O=C 3 NH 2 Citrulline + Pi

In the second step of the pathway the oxygen on the citrulline is replaced with a NH 3 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 − + H 3 N-C-H CH 2 NH + H 3 N-C-H + ATP AMP + PPi CH 2 COO − CH 2 COO CH 2 NH H-C-N =C O=C NH 2 COO NH 2 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 NH 3 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 CH H 2 O COO– Fumarate HO-C-H COO– NAD+ NADH C=O Glutamate -Kg + H 3 N-C-H CH 2 COO– 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 -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 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–Co. A • Acetoacetyl–Co. A • Pyruvate • α-Ketoglutarate • Succinyl–Co. A • 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 + NH 4+ – Transmination to oxaloacetate

α–Ketoglutarate Entry Point • Five carbon amino acids

α–Ketoglutarate Entry Point • Histidine

α–Ketoglutarate Entry Point • Proline and Arginine

Succinyl–Co. A Entry Point • Methionine, Valine & Isoleucine

Succinyl–Co. A 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 CO 2 e H 2 O 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-Co. A 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 (CH 3 COCH 3). 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-Co. A 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 H 2 O when the urea molecule is hydrolyzed free by the enzyme arginase. O + C H 2 N NH 2 – COO H 3 N-C-H CH 2 Urea CH 2 Citrulline + O HN=C NH NH 2 Ornithine After the urea is removed by hydrolysis, what remains is ornithine (click 1). Ornithine reacts with carbamoyl-PO 4 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-PO 4 that condenses with ornithine (click 1). As you can see below, carbamoyl-PO 4 is assembled from NH 4+ and CO 2 using 2 ATPs as an energy source and the enzyme carbamoyl-phosphate synthetase I. O NH 4+ + CO 2 + 2 ATP + 2 ADP + Pi C H 2 N OPO 3= COO- Carbamoyl-PO 4 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- + H 3 N-C-H CH 2 CH 2 NH CH 2 + NH O=C 3 NH 2 Citrulline + Pi

In the second step of the pathway the oxygen on the citrulline is replaced with a NH 3 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 − + H 3 N-C-H CH 2 NH + H 3 N-C-H + ATP AMP + PPi CH 2 COO − CH 2 COO CH 2 NH H-C-N =C O=C NH 2 COO NH 2 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 NH 3 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 CH H 2 O COO– Fumarate HO-C-H COO– NAD+ NADH C=O Glutamate -Kg + H 3 N-C-H CH 2 COO– 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 -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 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 + H 3 PO 4 D-glucose-6 -fosfato + H 2 O ΔG 0’ = +3, 3 kcal/mol ADP + H 3 PO 4 ATP + H 2 O ΔG 0’ = +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: ΔG 0’ = - 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 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.