Transcript Metabolismo dos aminoácidos e proteínas. II. Anabolismo

LICENCIATURA EM BIOLOGIA
DISCIPLINA
BIOQUÍMICA
Ano Lectivo de 2013/2014
Aula nº 24
21 MAI 2014
Ricardo Boavida Ferreira
Sala 40
Metabolismo dos aminoácidos e proteínas. II. Anabolismo
Assimilação do azoto e do enxofre (fundamentos).
Biossíntese dos aminoácidos proteicos. As famílias de aminoácidos. Aminoácidos essenciais,
não-essenciais e semi-essenciais. Breve referência à biossíntese dos aminoácidos raros das
proteínas e dos aminoácidoa não-proteicos.
Biossíntese de proteínas (fundamentos).
Material de estudo: diapositivos das aulas, bibliografia recomendada e textos de apoio.
Metabolismo dos
aminoácidos e proteínas
II. Anabolismo
ASSIMILAÇÃO DO AZOTO
Assimilação do carbono, do azoto e do enxofre
- Definição
- Quem é responsável?
- Assimilação do carbono:
Fotossíntese
CO2
Hidratos de carbono
- Assimilação do azoto:
N2
NO3-
Nitrogenase
Nitrato
redutase
NO2
Nitrito
- redutase
NH4+
NH4+
NH4+
Aminoácidos
NH4+
ESQUELETOS
CARBONADOS
- Assimilação do enxofre:
S2SO42-
Aminoácidos
Anaerobic
Most prevalent form
reduction
Nitrogen is a critical part of
amino acids, nucleotides
and other important
biomolecules.
Nome
Ião nitrato
Ião nitrito
Ião hiponitrito
Azoto (g)
Hidroxilamina
Amóniaco
estrutura
NO3NO2N2O22N2
NH2OH
NH3
nº oxidação do N
+5 (mais oxidada)
+3
+1
0
-1
-3 (mais reduzida)
Redução
Formas de azoto inorgânico disponíveis para plantas e
microrganismos autotróficos
Assimilação do azoto*: à semelhança da assimilação do carbono e do enxofre (e
contrariamente à do fósforo), é feita por plantas e microrganismos.
Principais formas de azoto inorgânico disponíveis para plantas e microrganismos: N2, NO3- e NH4+
N2 – Os organismos que fazem a fixação biológica do azoto contêm um compexo multienzimático – a
nitrogenase – capaz de quebrar a ligação covalente tripla que une os dois átomos de azoto do N2. Nas plantas
da família das Leguminosas, estabelece-se uma relação simbionte entre bactéria fixadora do azoto (do género
Rhizobium), que fornece NH4+ à planta, e a planta, que abastece a bactéria de fotoassimilados (i.e., hidratos de
carbono). A nitrogenase é inibida pelo O2, o que justifica a presença da legoglobina nos nódulos, que ficam,
assim, avermelhados. A parte proteica da legoglobina é fornecida pela planta e o respectivo grupo heme pela
bactéria simbionte.
NO3- - Trata-se de uma forma de azoto que pode ser absorvida e armazenada nos vacúolos das folhas das
plantas. A sua redução ao nível de amoníaco dá-se pela acção sequencial de duas importantes enzimas, que
consomem potencial redutor produzido pelas reacções fotoquímicas da fotossíntese: a nitrato redutase (NR),
que reduz o nitrato a nitrito, e a nitrito redutase (NiR), que reduz o nitrito a amoníaco.
NH4+ - Trata-se de uma forma tóxica de azoto, não podendo, por isso, acumular-se nas células (desacopla as
cadeias de transporte de electrões). O amónio é assimilado, isto é, incorporado em aminoácidos pelo
funcionamento do ciclo da glutamato sintase.
* Assimilação do azoto pode ser definida como o processo pelo qual o azoto passa de formas inorgânicas para
combinação orgânica.
Nitrogen fixation
Fixation of atmospheric N2
Certain free living bacteria
Symbiotic bacteria
Non-enzymatic (Haber method, 500 oC,
hundreds of Atms)
N2 + 3H2  2NH3
Enzymatic (nitrogenase complex, highly
conserved)
N2 + 10H+ + 8e- + 16ATP  2NH4+ + 16ADP + 16Pi + H2
1 e-/2ATP per cycle
2ATP binding shifts reduction potential
Fe-S clusters
Iron
Molybdenum
8 electrons (6 for N2, 2 for H2)
Mo, Fe S
homocitrate
Nitrogenase
Iron-molybdenum cofactor
ADP, Fe, S, MoFe
ADP, Fe, S, MoFe
Oxygen toxicity to bacterial nitrogenase: plant derived leghemoglobin
Reduced nitrogen (NH4+) via Glu and Gln
Gln synthetase (two step; key regulatory)
γ-Glu phosphate (mixed anhydride)
γ-Glu phosphate + NH4+ = Gln
Ala, Gly and others are partial/allosteric I
Primary
regulatory point
Gln synthetase
Adenylation, covalent, inhibitory
Adenylyltransferase, AT
Subunit structure of Gln synthetase (12 identical subunits, active sites)
Side view
Top view
AT = adenyltransferase
Uridylylation of Tyr (PII-UMP stimulates deadenylation)
Biossíntese dos aminoácidos
Amónio
+
Esqueleto carbonado
Vias de assimilação do amónio
Aminoácido
Biossíntese dos aminoácidos
Vias de assimilação do amónio
R
|
H2N –
C
|
COOH
Fornecido por metabolitos
das seguintes vias
metabólicas:
H
NH4+
Nitrito
redutase
- Glicólise;
- Via dos fosfatos de
pentose;
- Ciclo do ácido cítrico.
Nitrato
redutase
NO2N2
(atmosfera)
NO
Esqueleto carbonado:
NH4+ (solo)
-
(solo)
A via GS/GOGAT consome ATP
mas consegue assimilar o NH3
eficientemente quando a sua
concentração é baixa – isto porque
o valor do Km da enzima GS para o
NH3 é muito mais baixo que o da
enzima GDH.
Foi, por isso, sugerido que o ATP
gasto pela via GS/GOGAT é o preço
que as células que possuem esta via
têm de pagar para conseguirem
assimilar o NH3 presente em baixas
concentrações.
Ammonia Assimilation and Recycling
The glutamate synthase or GS-GOGAT cycle
For many years it was thought that bacteria and higher plants assimilate ammonia into glutamate via the GDH
pathway, as in certain fungi and yeasts. However, in bacteria it became clear in 1970 that an alternative pathway
of ammonia assimilation [involving glutamine synthetase (GS) [EC 6.3.1.2] and an NADPH-dependent
glutamine:2-oxoglutarate amidotransferase (GOGAT) [EC 1.4.1.13] or glutamate synthase, must be operating
when ammonia is present in the growth medium at low levels. Thus, N-starvation leads to derepression and
activation of GS (with a high affinity for NH3) and derepression of GOGAT, and repression of GDH (with a relatively
low affinity for NH3). High ammonia availability leads to repression and deactivation of GS and induction of GDH.
GDH
NH3 + 2-oxoglutarate + NADPH + H+ <---> glutamate + NADP+
GS-GOGAT
NH3 + glutamate + ATP ---> glutamine + ADP + Pi
glutamine + 2-oxoglutarate + NADPH + H+ ---> 2 glutamate + NADP+
Both the GDH and GS-GOGAT pathways produce 1 mole of glutamate from 1 mole each of NH3, 2-oxoglutarate
and NADPH. But note that the GS-GOGAT pathway is energetically more costly than the GDH pathway,
consuming 1 ATP.
Escherichia coli is now known to have two primary pathways for glutamate synthesis. The GS-GOGAT pathway is
essential for glutamate synthesis at low ammonium concentrations and for regulation of the glutamine pool, and is
used when the cell is not under energy limitation. The GDH pathway is used in glutamate synthesis when the cell
is limited for energy (and carbon; i.e. glucose-limited growth) but ammonium and phosphate are present in
excess. Synechocystis sp. strain PCC 6803 utilizes the GS-GOGAT pathway as the primary pathway of ammonia
assimilation, but the presence of GDH appears to offer a selective advantage for the cyanobacterium under
nonexponential growth conditions. These dual pathways may be common to bacteria, cyanobacteria, algae,
yeasts and fungi.
Re-examination of ammonia assimilation in yeasts and fungi now reveals the operation of alternative pathways of
glutamate synthesis, independent of NADPH-GDH:
Mutants of Aspergillus nidulans lacking NADP-GDH activity grow more poorly than wild-type strains on
ammonium as a sole nitrogen source. The leaky growth of these mutants is indicative of an alternative pathway
of ammonium assimilation and glutamate biosynthesis. A. nidulans mutants disrupted in the gltA encoding
GOGAT, were found to be dispensable for growth on ammonium in the presence of NADP-GDH. However, a
strain carrying the gltA inactivation together with an NADP-GDH structural gene mutation (gdhA) was unable to
grow on ammonium or on nitrogen sources metabolized via ammonium.
Schizosaccharomyces pombe mutants lacking either NADPH-GDH or GOGAT are still able to grow on
ammonium as sole nitrogen source. Complete lack of growth on ammonium as sole N source is seen only in
double mutants lacking both NADPH-GDH and GOGAT.
In contrast to Candida utilis, analysis of 15N-ammonium assimilation in actively growing mycelium of Agaricus
bisporus indicates participation of the GS-GOGAT pathway, and no participation of NADP-GDH. 13NH3 tracer
studies indicate that the GS-GOGAT pathway is the major route of ammonium assimilation in Candida albicans
and also in nitrogen-starved cultures of Saccharomyces cerevisiae and Candida tropicalis.
The yeast Saccharomyces cerevisiae synthesizes glutamate through the action of either NADP-glutamate
dehydrogenase (NADP-GDH), encoded by GDH1 (under conditions of ammonia excess), or through the
combined action of glutamine synthetase (GS) and glutamate synthase (GOGAT), encoded by GLN1 and GLT1
(under conditions of ammonia limitation). Dynamic modeling indicates that the GS-GOGAT pathway plays a
more important physiological role in yeast than is generally assumed. However, a double mutant of S. cerevisiae
lacking NADP-GDH and GOGAT activities was able to grow on ammonium as the sole nitrogen source and thus
to synthesize glutamate through a third pathway. A computer search for similarities between the GDH1
nucleotide sequence and the complete yeast genome led to the discovery that GDH1 showed high identity to an
open reading frame (GDH3) on chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 are strict
glutamate auxotrophs, indicating that GDH3 plays a significant physiological role, providing glutamate when
GDH1 and GLT1 are impaired. This appears to be the first example of a microorganism possessing three
pathways for glutamate biosynthesis.
Following the discovery of glutamate synthase (GOGAT) in bacteria, a similar activity was sought in plants. A
ferredoxin-dependent glutamate synthase [EC 1.4.7.1] was discovered in photosynthetic tissues of higher plants
in 1974, and an NADH-dependent "glutamate synthetase" in non-photosynthetic plant tissues in the same year.
O ciclo da glutamato sintase
Evidence in favor of the operation of the GS-GOGAT cycle as the primary pathway of ammonia assimilation in
higher plants includes:
- Almost complete inhibition of 15NH4+ assimilation by the glutamine synthetase (GS) inhibitor, methionine
sulfoximine (MSX).
- Quantitative analysis of 15NH4+ in Lemna minor is consistent with incorporation of 15N primarily into glutamineamide, followed by transfer to glutamate and the amino-group of glutamine via the action of GOGAT and GS,
respectively, provided that it is assumed that the GS-GOGAT cycle is compartmentilized in the chloroplast, and
that a second site of glutamine synthesis occurs in the cytoplasm.
- The maize gdh1-null mutant exhibits about 5% of the total GDH enzyme activity of wildtype plants. Although
this mutant exhibits a slightly reduced total rate of 15NH4+ assimilation, when methionine sulfoximine (MSX), a
potent inhibitor of GS is supplied, this completely blocks 15NH4+ assimilation in both the mutant and wildtype
roots and shoots. The contribution of GDH to net ammonia assimilation is small in comparison to that catalyzed
by the GS-GOGAT cycle.
- Mutants of Arabidopsis and barley defective in GS or GOGAT exhibit markedly impaired ammonia
assimilation, especially under photorespiratory conditions.
Enzyme kinetic considerations also suggest a role for the GS-GOGAT pathway in ammonia assimilation at low
tissue/cell ammonia concentrations. GS has a much higher affinity for ammonia than GDH and is viewed as a
scavenger of ammonia in bacteria and in plants.
The major role of GDH in tissue cultured cells is the oxidation of glutamate to provide sufficient carbon
skeletons for effective functioning of the TCA cycle.
In wildtype Arapidopsis, GDH1 mRNA accumulates to high levels in dark-adapted or sucrose-starved plants;
light or sucrose treatment each repress GDH1 mRNA accumulation. These results suggest that the GDH1 gene
product functions in the direction of glutamate catabolism under carbon-limiting conditions. Low levels of GDH1
mRNA present in leaves of light-grown plants can be induced by exogenously supplied ammonia. Under such
conditions of carbon and ammonia excess, GDH1 may function in the direction of glutamate biosynthesis. The
recessive Arabidopsis glutamate dehydrogenase-deficient mutant allele gdh1-1 cosegregates with the GDH1.
The gdh1-1 mutant displays a conditional phenotype; seedling growth is specifically retarded on media
containing exogenously supplied inorganic nitrogen, suggesting that GDH1 plays a nonredundant role in
ammonia assimilation under conditions of inorganic nitrogen excess. This is consistent with the fact that the
levels of mRNA for GDH1 and chloroplastic glutamine synthetase (GS2) are reciprocally regulated by light.
Aminoácidos essenciais
Os organismos diferem muito na sua capacidade de sintetizarem os aminoácidos de que necessitam para a
síntese proteica.
As plantas autotróficas e muitos microrganismos são auto-suficientes, no sentido em que conseguem
sintetizar os 20 aminoácidos proteicos.
A bactéria Leuconostoc mesenteroides consegue apenas sintetizar quatro aminoácidos proteicos, enquanto
que a bactéria Lactobacillus, que cresce no leite, não consegue sintetizar nenhum.
Os mamíferos estão numa posição intermédia, sendo capazes de sintetizar metade dos aminoácidos proteicos
– o que significa que têm de obter os outros na dieta alimentar.
Os aminoácidos que não podem ser sintetizados por um organismo em quantidade suficiente são
denominados aminoácidos essenciais ou indispensáveis e têm que ser fornecidos pela dieta alimentar.
Aqueles que podem ser sintetizados pelo organismo, a partir de precursores disponíveis, em quantidade
suficiente para satisfazer as suas necessidades são denominados não-essenciais ou dispensáveis.
São considerados aminoácidos essenciais para o homem a valina (Val), a leucina, (Leu), a isoloeucina (Ile) , o
triptofano (Trp), a fenilalanina (Phe), a lisina (Lys), a metionina (Met) e a treonina (Thr).
A arginina (Arg) e a histidina (His) são sintetizados em quantidade suficiente para satisfazer as necessidades
do homem adulto, mas não as de uma criança em crescimento. Estes aminoácidos são, por isso, designados
por semi- ou meio-essenciais.
A tirosina (Tyr) e a cisteína (Cys) são considerados não-essenciais se a dieta alimentar tiver quantidades
suficientes de fenilalanina e de metionina, respectivamente. Isto porque os mamíferos formam tirosina
directamente a partir da fenilalanina e porque a cisteína deriva o seu enxofre da metionina.
Em geral, os aminoácidos essenciais são aqueles com estruturas mais complicadas e formados por vias
metabólicas mais complexas, enquanto que os aminoácidos não-essenciais têm biossínteses mais
simples, a partir de precursores que estão normalmente presentes em todas as células.
A deficiência em um ou mais aminoácidos essenciais na dieta de um organismo origina, tipicamente,
um balanço de azoto negativo, isto é, o azoto total excretado pelo organismo excede o que é absorvido,
indicando degradação das proteínas dos tecidos para fornecer o (ou os) aminoácido que falta para a
síntese de novas proteínas prioritárias ou essenciais à sobrevivência do organismo. Os restantes
aminoácidos que compôem essas proteínas acumulam-se e sofrem catabolismo – daí a excreção do
azoto e o balanço de azoto negativo.
BIOSSÍNTESE DOS
AMINOÁCIDOS
5-phosphoribosyl-1-pyrophosphate (PRPP) from ribose phosphate
pyrophokinase: new intermediate in amino acid and nucleotide synthesis
Os 20 aminoácidos proteicos são agrupados em 6 famílias de acordo com os
metabolitos que lhes fornecem o esqueleto carbonado:
Metabolic precursor
organization
Glycolytic, citric acid and pentose phosphate
intermediates are the carbon skeleton sources
Glutamine and Glutamate are the N sources
20 common amino acid pathways (bacterial)
Biosynthetic Families
Metabolic Precursors
Amino Acids
α-Ketoglutarate
Glutamate, Glutamine, Proline, Arginine
3-Phosphoglycerate
Serine, Glycine, Cysteine
Oxaloacetate
Aspartate, Asparagine, Methionine, Threonine,
Lysine
Pyruvate
Alanine, Valine, Leucine, Isoleucine
Phosphoenolpyruvate
and erythrose 4-phosphate
Tryptophan, Phenlyalanine, Tyrosine
Ribose 5-phosphate
Histidine
The α-Ketoglutarate or Glutamate Family
-Ketoglutarate
Glutamate
Glutamine
Proline
Arginine
O 2-oxoglutarato, um intermediário do ciclo do ácido cítrico, é o precursor da
síntese do glutamato e dos outros membros desta família, a glutamina, a prolina, a
arginina e, nos fungos e em Euglena, a lisina.
Proline is cyclized Glutamate
Arg pathway missing in mammals
The 3-Phosphoglycerate or Serine Family
3-Phosphoglycerate
Serine
Glycine
Cysteine
O 3-fosfoglicerato, um intermediário da glicólise, é o precursor dos aminoácidos da
família da serina, que incluem, além deste aminoácido, a glicina e a cisteína.
Biosynthesis of serine/glycine
Biosynthesis of cysteine from serine in bacteria and plants:
A via da sulfidrilação directa
Mammals: methionine
and serine
Biosynthesis of
cysteine from
homocysteine and
serine in mammals:
A via da transulfuração
Assimilação do enxofre na natureza: é feita por plantas e microrganismos
A assimilação do enxofre na natureza, isto é, a incorporação do enxofre inorgânico no
aminoácido cisteína, é feita por plantas e microrganismos.
Há duas vias para a síntese de cisteína nos seres vivos:
- A via da sulfidrilação directa, em que plantas e microrganismos usam H2S para
sintetizar cisteína;
- A via da transulfuração, na qual os mamíferos utilizam o esqueleto carbonado da
serina e o enxofre da metionina para formar cisteína. Por este motivo, a metionina, mas
não a cisteína, é um aminoácido essencial para os mamíferos, homem incluído.
37
The Oxaloacetate or Aspartate Family
Oxaloacetate
Aspartate
Asparagine
Methionine
Lysine
Threonine
O oxaloacetato, um intermediário do ciclo do ácido cítrico, fornece o esqueleto
carbonado para a síntese dos seis aminoácidos da família do aspartato: aspartato,
asparagina, lisina (em bactérias e plantas, mas não em fungos), metionina, treonina
e isoleucina.
A isoleucina é, muitas vezes, incluída na família do piruvato, porque quatro das
cinco enzimas que participam na sua biossíntese são comuns às enzimas da via de
síntese da valina.
A metionina obtém o seu enxofre a partir da cisteína.
Aspartate to Lysine:
10 step synthesis
The Pyruvate Family
Pyruvate
Alanine
Valine
Leucine
Isoleucine
O piruvato, o produto final da glicólise, fornece os esqueletos carbonados para a
síntese da alanina e valina e quatro dos seis átomos de carbono da leucina.
Além disso, o piruvato contribui com dois carbonos para a síntese da isoleucina e,
em média, com 2,5 átomos para a síntese da lisina nas plantas e bactérias.
Como referido anteriormente, a isoleucina, um membro da família do aspartato, é
frequentemente incluída na família do piruvato.
The Phosphoenolpyruvate and Erythrose 4phosphate or Aromatic Family
Phosphoenolpyruvate
+
erythrose 4-phosphate
Chorismate
Tryptophan
Tyrosine
Fenilalanina, tirosina e triptofano são sintetizados a
partir do fosfoenolpiruvato, um intermediário da
glicólise, e da eritrose-4-fosfato, um intermediário da
via dos fosfatos de pentose.
Estes aminoácidos são sintetizados por uma via
metabólica ramificada divergente, em que o corismato
é o principal ponto de ramificação.
Phenylalanine
Chorismate: intermediate in aromatic amino acid
biosynthesis in bacteria and plants
O corismato é
sintetizado por uma
via composta por
sete reacções,
conhecida por via do
chiquimato, onde é
construído um anel
de benzeno.
EPSPS
Via do chiquimato:
Permite a síntese de um
anel benzénico (aromático)
Carbon from erythrose 4 phosphate
Carbon from phosphoenolpyruvate
Biosynthesis of Trp from
chorismate in bacteria and
plants
Biosynthesis of Phe and Tyr from
chorismate in bacteria and plants
involves prephenate
Animals (Phe
hydroxylation)
Nalguns organismos, incluindo o homem, a
tirosina pode ser sintetizada por hidroxilação
da fenilalanina, numa reacção irreversível
catalisada pela fenilalanina hidroxilase. Isto
explica que a tirosina não seja um aminoácido
essencial para o homem, uma vez que pode
ser formada a partir da fenilalanina que
ingerimos na dieta alimentar. Mas a
fenilalanina é um aminoácido essencial
porque a reacção catalisada pela fenilalanina
hidroxilase é irreversível, isto é, não podemos
formar fenilalanina a partir da tirosina.
O glifosato, um herbicida de grande sucesso
Glyphosate (N-(phosphonomethyl)glycine) is a non-selective systemic herbicide,
absorbed through the leaves, used to kill weeds, especially perennials. Some crops
have been genetically engineered to be resistant to it. Glyphosate was first sold by
Monsanto under the tradename Roundup, but is no longer under patent.
Glyphosate is an aminophosphonic analogue of the natural amino acid glycine and the
name is a contraction of glycine, phospho-, and -ate. Glyphosate was first discovered
to have herbicidal activity in 1970 by John Franz, while working for Monsanto.
Some microorganisms have a version of 5-enolpyruvoyl-shikimate-3-phosphate
synthetase (EPSPS) that is resistant to glyphosate inhibition. The version used in
genetically modified crops was isolated from Agrobacterium strain CP4 (CP4 EPSPS)
that was resistant to glyphosate. This CP4 EPSPS gene was cloned and transfected
into soybeans, and in 1996, such genetically modified soybeans were made
commercially available. This greatly improved the ability to control weeds in soybean
fields since glyphosate could be sprayed on fields without hurting the crop. As of 2005,
87% of U.S. soybean fields were planted with glyphosate resistant varieties.[
IUPAC name sodium 2[(hydroxy-oxidophosphoryl)methylamino]acetic
acid
Molecular
formula
C3H8NO5P
Molar mass
169.07 g mol-1
Aspecto de uma vinha localizada no vale do Tejo, ao lado de um terreno destinado à plantação de tomateiros e que
54 foi
pulverizado com glifosato num dia de vento. As plantas afectadas só recuperaram após terem sido pulverizadas com uma
solução aquosa de Phe + Tyr + Trp.
Mecanismo de acção do glifosato
Glyphosate kills plants by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the reaction of
shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phosphate (ESP). ESP is subsequently
dephosphorylated to chorismate an essential precursor in plants for the aromatic amino acids: phenylalanine, tyrosine and tryptophan.
The shikimate pathway is not present in animals, which obtain aromatic amino acids from their diet.
(5-enolpyruvylshikimate3-phosphate synthase)
Como nós não sintetizamos os
aminoácidos aromáticos (isto
é, não possuímos a enzima
EPSPS), o glifosato não
55 é tóxico
para nós.
Glyphosate and transgenic plants resistant to glyphosate
Glyphosate is a broad-spectrum herbicide that poisons many plant species so it is
frequently used to ‘burn down’ weeds on a field prior to the planting or emergence
of crops.
Before 1996, weeds were not observed to have evolved resistance to glyphosate in
the field, but since then, the introduction of transgenic glyphosate tolerant crops has
led to evolution of a number of resistant weeds as the result of the greatly
increased use of the herbicide particularly during the post-emergent growth of the
crops.
Glyphosate kills plants by interfering with the synthesis of the amino acids
phenylalanine, tyrosine and tryptophan. It does this by inhibiting the enzyme 5enolpyruvylshikimate-3-phosphate synthase (EPSPS).
Some microorganisms (e.g. Agrobacterium tumefaciens strain CP4) have a version
of EPSPS that is resistant to glyphosate inhibition. This version was expressed in
plants by genetic modification of crop plants which became resistant to glyphosate.
Genetic basis of glyphosate tolerance
The genetic basis of many of the glyphosate resistant weeds remains unknown; but
those studied in detail show that there is no single genetic alteration responsible in
all of the resistant weeds:
- Some plants evolved partial resistance to glyphosate through changes at amino
acid residue 106 of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)
gene. Mutations change amino acid 106 from proline to either serine or threonine,
results in an EPSPS weakly resistant to glyphosate.
- Other plants exhibit a reduced translocation of glyphosate to meristematic tissues.
- Yet other plants evolved EHSPS gene amplification. EPSPS enzyme activity from
resistant and susceptible plants was equally inhibited by glyphosate. Genomes of
resistant plants contained from 5-fold to more than 160-fold more copies of
the EPSPS gene than did genomes of susceptible plants. In these plants,
EPSPS expression was positively correlated with genomic EPSPS relative copy
number. The amplified genes were not clustered on the chromosomes but
distributed among all of the chromosomes.
- Other plants rapidly pumped the herbicide into vacuoles preventing contact of the
herbicide with the chloroplast.
The Ribose 5-Phosphate or Histidine Family
Regulação da biossíntese de
aminoácidos
Allosteric regulation of isoleucine biosynthesis
Great example of this regulation (feedback
inhibition)
Complex regulatory mechanisms in E.coli
(aspartate as source)
Note: isozymes (e.g., A1, B2) some with (e.g., C1)
and some without (e.g., C2) allosteric regulation
Além de oligopéptidos, polipéptidos e proteínas, os aminoácidos são precursores
de uma grande variedade de compostos de grande importância biológica
Synthesis of Biologically Important Compounds
In addition to their role in protein synthesis, energy production and gluconeogenesis, many
amino acids serve as precursors for the synthesis of other amino acids and other biologically
important cornpounds. Many oligopeptides containing up to 20 residues, including hormones,
antibiotics and antitumour agents, are synthesized in living organisms by mechanisms
different from the usual ribosome-dependent processes of protein synthesis. The dipeptides
carnosine (β-alanylhistidine) and anserine (β-alanyl-1-N-methylhistidine) are synthesized
enzymatically from β-alanine and histidine, and from carnosine and S-adenosylmethionine,
respectively.
Glutathione (γ-glutamylcysteinylglycine) plays a variety of roles in living organisms. This
tripeptide is synthesized by a two-step enzymatic pathway: (1) the formation of a peptide
linkage between the γ-carboxyl group of glutamate and the amino group of cysteine, to produce
γ-glutamylcysteine; (2) the condensation of this dipeptide with glycine, to form glutathione. Thus
the order of the amino acids in glutathione is specifically determined by the enzymes catalysing
the formation of each peptide bond.
At least 90 different peptide antibiotics are produced by strains of Bacillus subtilis and B. breuis.
Gramicidin S, for example, is a cyclic decapeptide composed of two identical pentapeptides
(D-phenylalanine-L-proline-L-valine-L-ornithine-L-leucine). Gramicidin S is synthesized by a
multienzyme complex, gramicidin synthetase, composed of two enzyrnes, one of which, serving
as a template, specifies the amino acid sequence in the antibiotic.
S-Adenosylmethionine (SAM), the metabolically activated form of methionine, functions as
an important source of methyl and propylamino groups for a wide variety of compounds,
including alkaloids, choline, creatine, adrenaline, N-methylated amino acids, nucleotides,
and polyamines, as well as for phospholipids, proteins, polysaccharides, and nucleic
acids, It is synthesized from methionine and ATP, in a reaction catalysed by SAM synthase.
A wide variety of amines occurring in bacteria, plants and animais are derived directly or
indirectly from amino acids by decarboxylation; these include ethylamine (from alanine),
agmatine (from arginine), γ-aminobutyric acid (from glutamate), methylamine (from
glycine), histamine (from histidine), cadaverine (from Iysine), putrescine (from ornithine),
phenylethylamine (from phenylalanine), ethanolamine (from serine), tryptamine and 5hydroxytryptamine (or serotonin; from tryptophan), and tyramine and dopamine (from
tyrosine). These amines and their derivatives often play a variety of physiologically important
roles. For example, γ-aminobutyric acid, phenylethylamine, tryptamine, 5-hydroxytryptamine,
or serotonin, tyramine, dopamine, noradrenaline, and adrenaline are all neurologically active
compounds, whereas histamine, a powerful vasodilator, is involved in allergic reactions.
Tyrosine plays severaI important roles in animal metabolism as a precursor to melanins,
thyroid hormones (thyroxine and triiodothyronine), and catecholamines (dopamine,
noradrenaline and adrenaline). In the synthesis of melanins, tyrosinase catalyses first the
hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (dopa), followed by the oxidation of
dopa to phenylalanine- 3,4-quinone (dopaquinone). Dopaquinone undergoes a sequence of
reactions, including polymerization, to yield both red and black melanins.
Another enzyme forming dopa is tyrosine hydroxylase, which catalyses the first reaction in the
sequential enzymatic pathway leading to the biosynthesis of catecholamines. Dopa is then
decarboxylated to yield 3,4-dihydroxyphenylethylamine (dopamine). Dopamine is hydroxylated
to norepinephrine (noradrenaline), which in turn is methylated by SAM to give epinephrine
(adrenaline).
Putrescine, or 1,4-diaminebutane, is synthesized by decarboxylation of ornithine, in a reaction
catalysed by ornithine decarboxylase, a highly regulated enzyme. Another route to putrescine
formation involves the conversion of arginine to agmatine by arginine decarboxylase, followed
by cleavage of agmatine to putrescine and urea by agmatine ureohydrolase. Putrescine is an
intermediate in the biosynthesis of two important polyamines, spermidine and spermine.
Spermidine is synthesized enzymatically by the SAM-mediated transfer of a propylamino group
to putrescine. The enzymatic transfer of an additional propylamino group from SAM to
spermidine produces spermine. These polycations play multiple roles in stabilizing negatively
charged intracellular components such as nucleic acids and membranes.
Creatine phosphate, which serves as a source of high energy phosphate in mammalian
muscle and brain, is synthesized in three steps from arginine, glycine, and methionine.
There are four classes of tetrapyrrole compounds, haems, chlorophylls, phicobilins and
cobalamins, all of which are synthesized from a common precursor, 6-aminolevulinic acid
(ALA). In bacteria and animais, ALA is synthesized by the condensation of glycine and succinylCoA, with loss of carbon dioxide, in a reaction catalysed by ALA synthase. ln plants, however,
ALA is formed from glutamate by a three-step pathway.
An enorrnous amount of carbon in the biosphere passes through the pathway leading to lignin
biosynthesis, the major constituent of woody tissue. ln the first reaction, phenylalanine
ammonia Iyase catalyses the cleavage of phenylalanine to trans-cinnamic acid and NH4+.
Cinnamic acid is a precursor for the synthesis of a huge number of plant substances, including
lignin, tannins, flavonoids, pigments, many of the flavour components of spices, and various
alkaloids, such as morphine and colchicine.
ln addition, the synthesis of a variety of other important molecules utilizes various amino acids
as precursors. Thus β-alanine is a component of CoA, asparagine is a major form of transport
of organic nitrogenous compounds in plants, and aspartate is involved in purine and pyrimidine
biosynthesis. Glutamate is a precursor of folic acid; glutamine contributes to the synthesis of a
variety of substances, including purines, pyrimidines, ATP, cytidine triphosphate (CTP),
NAD, amino sugars, and glycoproteins; cystein is a precursor of taurine, isethionic acid,
CoA, vasopressin, various types of pigments, including phaeomaline and trichochromes,
and other sulphur-containing compounds. Glycine also plays multiple roles, including
contributions to the one-carbon pool and as a precursor of purines, glyoxylate, and various
conjugates such as hippurate and glycocholate. Histidine is involved in ergothionine and
homocarnosine biosynthesis, and methionine, via SAM, is the precursor of the, plant hormone
ethylene, which influences plant growth and development and induces the ripening of fruits.
Serine is involved in the biosynthesis of phospholipids, and tryptophan is the precursor of
several important physiological substances, including NAD, NADP, and the plant hormone
indole 3-acetic acid.
Questões
1. Defina os seguintes conceitos:
a) Turnover de proteínas.
b) Endopeptidase.
c) Ubiquitina.
d) Carbamoíl-fosfato.
e) Aminoácido cetogénico.
f) Aminoácido essencial.
g) Inibição por feedback.
2. Justifique as seguintes afirmações:
a) No caso do homem, a fenilalanina é considerada um aminoácido
essencial, mas não a tirosina.
b) A hidrólise de proteínas é um processo exergónico:
Polipéptido + (n-1)H2O  naminoácidos
ΔGO’ < 0
Explique, sucintamente, porque razão as células gastam energia
para degradar as suas proteínas.
c) O ciclo da ureia desempenha um papel essencial no metabolismo do
azoto do homem.
d) O glifosato, o herbicida descoberto há 40 anos e ainda o mais utilizado
à escala mundial, é letal para as plantas, mas não para o
homem.
3. Considere a assimilação do carbono, azoto e enxofre e a biossíntese dos aminoácidos.
3.1 - Defina metabolismo do carbono, do azoto e do enxofre.
3.2 – Defina, para cada um, a forma inorgânica que é assimilada, o tipo de composto orgânico formado, a
via enzimática envolvida e os organismos em que ocorre.
3.3 – Que vias metabólicas fornecem os esqueletos carbonados para a síntese dos aminoácidos?
3.4 – Identifique as seis famílias biossintéticas de aminoácidos.
3.5 – Defina, para o caso do Homem, aminoácidos não-essenciais, semi-essenciais e essenciais, dando
dois exemplos de cada.
4 - Considere o metabolismo do azoto e dos aminoácidos. Identifique o significado das letras (nomes de
enzimas, de complexos enzimáticos ou de vias metabólicas) e dos números (nomes de compostos, de
famílias de compostos ou de espécies animais) na figura seguinte:
Legenda: A B C D E
F G H I J K 123–
4–
5–
6–
7–
8–
9–
10 –
11 –
12 –
13 –
14 –
15 –
16 –
17 -
FIM