CO2 TAG’ SÍNTESIS’Y’DEGRADACION’ … · The fidelity of protein synthesis requires accurate recognition of three-base codons on messenger RNA. Recall that the genetic code

SÍNTESIS Y DEGRADACION DE PROTEÍNAS

AMINOÁCIDOS CETOÁCIDOS PROTEÍNAS

GLUCOSA

NH4+

NH2-‐CO-‐NH2 ÁCIDO ÚRICO

(Aves)

Síntesis

Degradación

ACoA AGCL

CO2

Oxidación

N

RECAMBIO PROTEICO (PROTEIN TURNOVER)

TAG

N

PROTEÍNAS DIETARIAS


PROTEÍNAS

Síntesis (S)

Degradación (D)

AMINOÁCIDOS

La síntesis y la degradación de proteínas son las dos caras de un proceso que ocurre en forma conSnua en todos los tejidos del organismo y que llamamos recambio proteico (protein turnover).

El resultado del balance de ambos procesos determina si aumenta o dismuniye la canSdad de proteínas corporales.

S > D

PROTEÍNAS

S < D PROTEÍNAS

PROTEÍNAS S = D

2-‐8 % / día

PROTEÍNAS

Síntesis (S)

Degradación (D)

AMINOÁCIDOS

2520 g/ d

Novillo de 300 kg que gana 1kg/d peso vivo

2680 g/ d

Retenido

160 g /d

PROTEÍNAS

Síntesis

Degradación

AMINOÁCIDOS

SÍNTESIS DE PROTEÍNAS

ENZIMAS (CITOSÓLICAS, DE ORGANELAS, NUCLEARES)

DE MEMBRANA (RECEPTORES, CANALES, TRANSPORTADORES) MICROTÚBULOS Y MICROFILAMENTOS

CONSTITUTIVAS

EXPORTABLES PROTEÍNAS PLASMÁTICAS

PROTEÍNAS DE LA LECHE

PROTEÍNAS DE PELOS, FIBRAS Y UÑAS

HISTONAS

COSTO 4 ATP (122 kJ o 29 kcal )/ Mol aminoácidos

SE DEGRADAN EN EL CUERPO

NO SE DEGRADAN EN EL CUERPO: LOS AMINOÁCIDOS SE PIERDEN

QUERATINA DE PIEL Y MUCOSAS

ENZIMAS DIGESTIVAS

PROTEÍNAS DEL FETO Y PLACENTA FETAL

HORMONAS

89530.1 Basics of Translation

10 2 4 to produce the larger proteins effectively. Lower error frequencies are conceivable; however, except for the largest proteins, they will not dramati-cally increase the percentage of proteins with accurate sequences. In addition, such lower error rates are likely to be possible only by a reduction in the rate of protein synthesis because additional time for proofreading is required. In fact, the observed values of ́ are close to 10 2 4 . An error frequency of about 10 ! 4 per amino acid residue was selected in the course of evolution to accu-rately produce proteins consisting of as many as 1000 amino acids while maintaining a remarkably rapid rate for protein synthesis.

Transfer RNA molecules have a common design

The fidelity of protein synthesis requires accurate recognition of three-base codons on messenger RNA. Recall that the genetic code relates each amino acid to a three-letter codon (Section 4.6). An amino acid cannot itself recog-nize a codon. Consequently, an amino acid is attached to a specific tRNA molecule that recognizes the codon by Watson–Crick base-pairing. Transfer RNA serves as the adapter molecule that binds to a specific codon and brings with it an amino acid for incorporation into the polypeptide chain.

Consider yeast alanyl-tRNA, so called because it will carry the amino acid alanine. This adapter molecule is a single chain of 76 ribonucleotides (Figure 30.2). The 5 9 terminus is phosphorylated (pG), whereas the 3 9 ter-minus has a free hydroxyl group. The amino acid-attachment site is the 3 9 -hydroxyl group of the adenosine residue at the 3 9 terminus of the mole-cule. The sequence 5 9 -IGC-3 9 in the middle of the molecule is the anticodon, where I is the purine base inosine . It is complementary to 5 9 -GCC-3 9 , one of the codons for alanine.

Thousands of tRNA sequences are known. The striking finding is that all of them can be arranged in a cloverleaf pattern in which about half the residues are base-paired (Figure 30.3). Hence, tRNA molecules have many common structural features. This finding is not unexpected, because all tRNA molecules must be able to interact in nearly the same way with the ribosomes, mRNAs, and protein factors that participate in translation.

Codon

Anticodon

C G I

G C C

3! 5!

5! 3!

O

HN

N N

N

riboseInosine

U

U

CCA

3!

5! p

OHAmino acid-attachment site

Phosphorylated5! terminus

GG

C

CG T "

A

Anticodonloop

"Extra arm"(variable)

DHU loopT"C loop

UH2

FIGURE 30.3 General structure of tRNA molecules. Comparison of the base sequences of many tRNAs reveals a number of conserved features.

G CU

U

UH2

UH2

UH2

m2G

mG

U

UU

UG CC GG UG CG C

C GC

C

GC G

G

U A

AC G G G

ACCA

3!

5! p

OH Amino acid-attachment site

GCC GG

GG

UC

C

U UCGGA

GG

GCCCCGCG

A

T "

"

Iml

A

A

Anticodon

FIGURE 30.2 Alanyl-tRNA sequence. The base sequence of yeast alanyl-tRNA and the deduced cloverleaf secondary structure are shown. Modified nucleosides are abbreviated as follows: methylinosine (mI), dihydrouridine (UH2), ribothymidine (T), pseudouridine (c), methylguanosine (mG), and (dimethylguanosine (m2G). Inosine (I), another modified nucleoside, is part of the anticodon.

89930.2 Aminoacyl-tRNA Synthetases

This activated species is a mixed anhydride in which the carboxyl group of the amino acid is linked to the phosphoryl group of AMP; hence, it is also known as aminoacyl-AMP .

Aminoacyl adenylate

O

+H3N P

O

HR

O O–

Oadenine

OH

O

HO

The next step is the transfer of the aminoacyl group of aminoacyl-AMP to a particular tRNA molecule to form aminoacyl-tRNA .

Aminoacyl-AMP 1 tRNA ! aminoacyl-tRNA 1 AMP

The sum of these activation and transfer steps is

Amino acid 1 ATP 1 tRNA ! aminoacyl-tRNA 1 AMP 1 PPi

The D G 8 9 of this reaction is close to 0, because the free energy of hydrol-ysis of the ester bond of aminoacyl-tRNA is similar to that for the hydrolysis of ATP to AMP and PP i . As we have seen many times, the reaction is driven by the hydrolysis of pyrophosphate. The sum of these three reactions is highly exergonic:

Amino acid 1 ATP 1 tRNA 1 H2O ¡aminoacyl-tRNA 1 AMP 1 2 Pi

Thus, the equivalent of two molecules of ATP is consumed in the synthesis of each aminoacyl-tRNA . One of them is consumed in forming the ester link-age of aminoacyl-tRNA, whereas the other is consumed in driving the reac-tion forward.

The activation and transfer steps for a particular amino acid are cata-lyzed by the same aminoacyl-tRNA synthetase. Indeed, the aminoacyl-AMP intermediate does not dissociate from the synthetase . Rather, it is tightly bound to the active site of the enzyme by noncovalent interactions.

We have already encountered an acyl adenylate intermediate in fatty acid activation (Section 22.2). The major difference between these reactions is that the acceptor of the acyl group is CoA in fatty acid activation and tRNA in amino acid activation. The energetics of these biosyntheses are very simi-lar: both are made irreversible by the hydrolysis of pyrophosphate.

Aminoacyl-tRNA synthetases have highly discriminating amino acid activation sites

Each aminoacyl-tRNA synthetase is highly specific for a given amino acid. Indeed, a synthetase will incorporate the incorrect amino acid only once in 10 4 or 10 5 reactions. How is this level of specificity achieved? Each amino-acyl-tRNA synthetase takes advantage of the properties of its amino acid substrate. Let us consider the challenge faced by threonyl-tRNA synthe-tase. Threonine is especially similar to two other amino acids—namely, valine and serine. Valine has almost exactly the same shape as that of threo-nine, except that valine has a methyl group in place of a hydroxyl group. Serine has a hydroxyl group, as does threonine, but lacks the methyl group. How can the threonyl-tRNA synthetase avoid coupling these incorrect amino acids to threonyl-tRNA?

R AMP

tRNAO

+H3N

O

R H

O

Acyl adenylateintermediate

Fatty acyl CoA

Aminoacyl-tRNA

CoA

H2

H3CC S

O

( (n

+H3N

+H3N

+H3N COO–

HC

HO CH3

H

COO–

HC

HO

COO–

HC

H3C CH3

H

Threonine

Valine

Serine

HH

903

species. A ribosome contains one copy of each RNA molecule, two copies each of the L7 and L12 proteins, and one copy of each of the other proteins. The L7 protein is identical with L12 except that its amino terminus is acety-lated (Section 10.3). Both the 30S and the 50S subunits can be reconstituted in vitro from their constituent proteins and RNA. This reconstitution is an outstanding example of the principle that supramolecular complexes can form spontaneously from their macromolecular constituents .

The structures of both the 30S and the 50S subunits as well as the com-plete 70S ribosome have been determined (Figure 30.14). The features of these structures are in remarkable agreement with interpretations of less-direct experimental probes. These structures provide an invaluable frame-work for examining the mechanism of protein synthesis.

Ribosomal RNAs (5S, 16S, and 23S rRNA) play a central role in protein synthesis

The prefix ribo in the name ribosome is apt because RNA constitutes nearly two-thirds of the mass of these large molecular assemblies. The three RNAs present—5S, 16S, and 23S—are critical for ribosomal architecture and func-tion. They are formed by the cleavage of primary 30S transcripts and further processing. These molecules fold into structures that allow them to form internal base pairs. Their base-pairing patterns were deduced by comparing the nucleotide sequences of many species to detect conserved sequences as well as conserved base pairings. For instance, the 16S RNA of one species may have a G–C base pair, whereas another may have an A–U base pair, but the location of the base pair is the same in both molecules. Chemical modi-fication and digestion experiments supported the structures deduced from sequence comparisons (Figure 30.15). The striking finding is that across all species, ribosomal RNAs (rRNAs) are folded into defined structures that have many short duplex regions .

For many years, ribosomal proteins were presumed to orchestrate pro-tein synthesis and ribosomal RNAs were presumed to serve primarily

as structural scaffolding. The current view is almost the reverse. The discovery of catalytic RNA (Section 29.4) made biochemists receptive to the possibility that RNA plays a much more active role in ribosomal function. The detailed structures make it clear that the key sites in the ribosome, such as those that catalyze the formation of the peptide bond and interact with mRNA and tRNA, are composed almost entirely of RNA. Contributions from the pro-teins are minor. The almost inescapable conclusion is that the ribosome ini-tially consisted only of RNA and that the proteins were added later to fine-tune

70S ribosome 30S subunit50S subunit

FIGURE 30.14 The ribosome at high resolution. Detailed models of the ribosome based on the results of x-ray crystallographic studies of the 70S ribosome and the 30S and 50S subunits: (left) view of the part of the 50S subunit that interacts with the 30S subunit; (center) side view of the 70S ribosome; (right) view of the part of the 30S subunit that interacts with the 50S subunit. 23S RNA is shown in yellow, 5S RNA in orange, 16S RNA in green, proteins of the 50S subunit in red, and proteins of the 30S subunit in blue. Notice that the interface between the 50S and the 30S subunits consists entirely of RNA. [Drawn from 1GIX. pdb and 1GIY.pdb.]

tRNA

Aminoácidos

Ribosoma

905

its functional properties. This conclusion has the pleasing consequence of dodging a “chicken and egg” question: How can complex proteins be synthe-sized if complex proteins are required for protein synthesis?

Ribosomes have three tRNA-binding sites that bridge the 30S and 50S subunits

Three tRNA-binding sites in ribosomes are arranged to allow the formation of peptide bonds between amino acids encoded by the codons on mRNA (Figure 30.16). The mRNA fragment being translated at a given moment is bound within the 30S subunit. Each of the tRNA molecules is in contact with both the 30S subunit and the 50S subunit. At the 30S end, two of the three tRNA molecules are bound to the mRNA through anticodon–codon base pairs. These binding sites are called the A site (for a minoacyl) and the P site (for p eptidyl). The third tRNA molecule is bound to an adjacent site called the E site (for e xit).

The other end of each tRNA molecule, the end without the anticodon, interacts with the 50S subunit. The acceptor stems of the tRNA molecules occupying the A site and the P site converge at a site where a peptide bond is formed. A tunnel connects this site to the back of the ribosome, through which the polypeptide chain passes during synthesis (Figure 30.17).

The start signal is usually AUG preceded by several bases that pair with 16S rRNA

How does protein synthesis start? The simplest possibility would be for the first 3 nucleotides of each mRNA to serve as the first codon; no special start signal would then be needed. However, experiments show that translation in bacteria does not begin immediately at the 5 9 terminus of mRNA. Indeed, the first translated codon is nearly always more than 25 nucleotides away from the 5 9 end. Furthermore, in bacteria, many mRNA molecules are polycistronic —that is, they encode two or more polypeptide chains. For example, a single mRNA molecule about 7000-nucleotides long specifies five enzymes in the biosynthetic pathway for tryptophan in E. coli. Each of these five proteins has its own start and stop signals on the mRNA. In fact, all known mRNA molecules contain signals that define the beginning and end of each encoded polypeptide chain .

A clue to the mechanism of initiation was the finding that nearly half the amino-terminal residues of proteins in E. coli are methionine. In fact, the

FIGURE 30.16 Transfer RNA-binding sites. (A) Three tRNA-binding sites are present on the 70S ribosome. They are called the A (for aminoacyl), P (for peptidyl), and E (for exit) sites. Each tRNA molecule contacts both the 30S and the 50S subunit. (B) The tRNA molecules in sites A and P are base-paired with mRNA. [(B) Drawn from 1JGP. pdb.]

A siteP siteE sitemRNA

A siteP siteE site

(A) (B)

FIGURE 30.17 An active ribosome. This schematic representation shows the relations among the key components of the translation machinery.

5’ 3’

Polypeptidechannel

mRNA

E

30S

50S

E AP

A

mRNA

SiSo E SiSo P SiSo A PepSdil tRNA Aminoacil tRNA Exit (salida)

SUSTRATOS

Stryer, 2015

¿QUÉ NECESITAMOS PARA LA SÍNTESIS?







Codon

Anticodon

C G I

G C C

3! 5!

5! 3!

O

HN

N N

N

riboseInosine

U

U

CCA

3!

5! p



GG

C

CG T "

A

Anticodonloop


DHU loopT"C loop

UH2


G CU

U

UH2

UH2

UH2

m2G

mG

U

UU

UG CC GG UG CG C

C GC

C

GC G

G

U A

AC G G G

ACCA

3!

5! p


GCC GG

GG

UC

C

U UCGGA

GG

GCCCCGCG

A

T "

"

Iml

A

A

Anticodon




Aminoacyl adenylate

O

+H3N P

O

HR

O O–

Oadenine

OH

O

HO












R AMP

tRNAO

+H3N

O

R H

O


Fatty acyl CoA

Aminoacyl-tRNA

CoA

H2

H3CC S

O

( (n

+H3N

+H3N

+H3N COO–

HC

HO CH3

H

COO–

HC

HO

COO–

HC

H3C CH3

H

Threonine

Valine

Serine

HH



Aminoacyl adenylate

O

+H3N P

O

HR

O O–

Oadenine

OH

O

HO












R AMP

tRNAO

+H3N

O

R H

O


Fatty acyl CoA

Aminoacyl-tRNA

CoA

H2

H3CC S

O

( (n

+H3N

+H3N

+H3N COO–

HC

HO CH3

H

COO–

HC

HO

COO–

HC

H3C CH3

H

Threonine

Valine

Serine

HH







Codon

Anticodon

C G I

G C C

3! 5!

5! 3!

O

HN

N N

N

riboseInosine

U

U

CCA

3!

5! p



GG

C

CG T "

A

Anticodonloop


DHU loopT"C loop

UH2


G CU

U

UH2

UH2

UH2

m2G

mG

U

UU

UG CC GG UG CG C

C GC

C

GC G

G

U A

AC G G G

ACCA

3!

5! p


GCC GG

GG

UC

C

U UCGGA

GG

GCCCCGCG

A

T "

"

Iml

A

A

Anticodon


ATP AMP+2Pi

Aminoacil-tRNA sintetasa

Aminoácido

tRNA

Aminoacil-‐tRNA

(aminoácido específica)

SÍNTESIS DE PROTEÍNAS

Stryer, 2015

905

its functional properties. This conclusion has the pleasing consequence of dodging a “chicken and egg” question: How can complex proteins be synthe-sized if complex proteins are required for protein synthesis?

Ribosomes have three tRNA-binding sites that bridge the 30S and 50S subunits

Three tRNA-binding sites in ribosomes are arranged to allow the formation of peptide bonds between amino acids encoded by the codons on mRNA (Figure 30.16). The mRNA fragment being translated at a given moment is bound within the 30S subunit. Each of the tRNA molecules is in contact with both the 30S subunit and the 50S subunit. At the 30S end, two of the three tRNA molecules are bound to the mRNA through anticodon–codon base pairs. These binding sites are called the A site (for a minoacyl) and the P site (for p eptidyl). The third tRNA molecule is bound to an adjacent site called the E site (for e xit).

The other end of each tRNA molecule, the end without the anticodon, interacts with the 50S subunit. The acceptor stems of the tRNA molecules occupying the A site and the P site converge at a site where a peptide bond is formed. A tunnel connects this site to the back of the ribosome, through which the polypeptide chain passes during synthesis (Figure 30.17).

The start signal is usually AUG preceded by several bases that pair with 16S rRNA

How does protein synthesis start? The simplest possibility would be for the first 3 nucleotides of each mRNA to serve as the first codon; no special start signal would then be needed. However, experiments show that translation in bacteria does not begin immediately at the 5 9 terminus of mRNA. Indeed, the first translated codon is nearly always more than 25 nucleotides away from the 5 9 end. Furthermore, in bacteria, many mRNA molecules are polycistronic —that is, they encode two or more polypeptide chains. For example, a single mRNA molecule about 7000-nucleotides long specifies five enzymes in the biosynthetic pathway for tryptophan in E. coli. Each of these five proteins has its own start and stop signals on the mRNA. In fact, all known mRNA molecules contain signals that define the beginning and end of each encoded polypeptide chain .

A clue to the mechanism of initiation was the finding that nearly half the amino-terminal residues of proteins in E. coli are methionine. In fact, the

FIGURE 30.16 Transfer RNA-binding sites. (A) Three tRNA-binding sites are present on the 70S ribosome. They are called the A (for aminoacyl), P (for peptidyl), and E (for exit) sites. Each tRNA molecule contacts both the 30S and the 50S subunit. (B) The tRNA molecules in sites A and P are base-paired with mRNA. [(B) Drawn from 1JGP. pdb.]

A siteP siteE sitemRNA

A siteP siteE site

(A) (B)

FIGURE 30.17 An active ribosome. This schematic representation shows the relations among the key components of the translation machinery.

5’ 3’

Polypeptidechannel

mRNA

E

30S

50S

E AP

A

RIBOSOMA

Canal polipendico

siSo E: Exit (salida)

siSo P: PepSdil tRNA

siSo A: Aminoacil tRNA







Codon

Anticodon

C G I

G C C

3! 5!

5! 3!

O

HN

N N

N

riboseInosine

U

U

CCA

3!

5! p



GG

C

CG T "

A

Anticodonloop


DHU loopT"C loop

UH2


G CU

U

UH2

UH2

UH2

m2G

mG

U

UU

UG CC GG UG CG C

C GC

C

GC G

G

U A

AC G G G

ACCA

3!

5! p


GCC GG

GG

UC

C

U UCGGA

GG

GCCCCGCG

A

T "

"

Iml

A

A

Anticodon




Aminoacyl adenylate

O

+H3N P

O

HR

O O–

Oadenine

OH

O

HO












R AMP

tRNAO

+H3N

O

R H

O


Fatty acyl CoA

Aminoacyl-tRNA

CoA

H2

H3CC S

O

( (n

+H3N

+H3N

+H3N COO–

HC

HO CH3

H

COO–

HC

HO

COO–

HC

H3C CH3

H

Threonine

Valine

Serine

HH

Stryer, 2015

90930.3 The Ribosome

of the transition state that follows the attack is not established and several models are plausible. One model proposes roles for the 2 9 OH of the adeno-sine of the tRNA in the P site and a molecule of water at the peptidyl trans-ferase center (Figure 30.22B). The nucleophilic attack of the a -amino group generates an eight-membered transition state in which three protons are shuttled about in a concerted manner. The proton of the attacking amino group hydrogen bonds to the 2 9 oxygen of ribose of the tRNA. The hydrogen of 2 9 OH in turn interacts with the oxygen of the water molecule at the center, which then donates a proton to the carbonyl oxygen. Collapse of the transi-tion state with the formation of the peptide bond allows protonation of the 3 9 OH of the now empty tRNA in the P site (Figure 30.22C). The stage is now set for translocation and formation of the next peptide bond.

The formation of a peptide bond is followed by the GTP-driven translocation of tRNAs and mRNA

With the formation of the peptide bond, the peptide chain is now attached to the tRNA whose anticodon is in the A site on the 30S subunit. The two subunits rotate with respect to one another, and this structural change places the CCA end of the same tRNA and its peptide in the P site of the large subunit (Figure 30.23). Another aminoacyl-tRNA arrives and binds at the A site (1). Again, peptide bond synthesis occurs (2). However, protein synthesis cannot continue without the translocation of the mRNA and the tRNAs within the ribosome. Elongation factor G (EF-G, also called translo-case ) catalyzes the movement of mRNA, at the expense of GTP hydrolysis, by a distance of three nucleotides. Now, the next codon is positioned in the A site for interaction with the incoming aminoacyl-tRNA (3). The pepti-dyl-tRNA moves out of the A site into the P site on the 30S subunit and at the same time, the deacylated tRNA moves out of the P site into the E site and is subsequently released from the ribosome (4). The movement of the peptidyl-tRNA into the P site shifts the mRNA by one codon, exposing the next codon to be translated in the A site.

APE

AP

E

Tunnel

30S

50S

Aminoacyl-tRNAbinding

5’ 3’

5’ 3’ 5’ 3’

5’ 3’ 5’ 3’Peptide-bond

formation

Translocation

GTP

GDP + Pi

Elongationfactor G

tRNAdissociation

1 2

4

3

FIGURE 30.23 Mechanism of protein synthesis. The cycle begins with peptidyl-tRNA in the P site. (1) An aminoacyl-tRNA binds in the A site. (2) With both sites occupied, a new peptide bond is formed. (3) The tRNAs and the mRNA are translocated through the action of elongation factor G, which moves the deacylated tRNA to the E site. (4) Once there, the tRNA is free to dissociate to complete the cycle.

Unión del aminoacil-tRNA

Formación de la unión peptídica

Translocación

Disociación de t-RNA

Stryer, 2015

GTP

GDP + Pi

GTP GDP + Pi

911

A third factor, RF3, another GTPase, catalyzes the removal of RF1 or RF2 from the ribosome upon release of the newly synthesized protein.

RF1 and RF2 are compact proteins that, in eukaryotes, resemble a tRNA molecule. When bound to the ribosome, the proteins unfold to bridge the gap between the stop codon on the mRNA and the peptidyl transferase center on the 50S subunit (Figure 30.26). The RF interacts with the peptidyl transferase center using a loop containing a highly conserved glycine-glycine-glutamine (GGQ) sequence, with the glutamine methylated on the amide nitrogen atom of the R group. This modified glutamine (assisted by the peptidyl transferase) is crucial in promoting a water molecule 9 s attack on the ester linkage between the tRNA and the polypeptide chain, freeing the polypeptide chain. The detached polypeptide leaves the ribosome. Transfer RNA and messenger RNA remain briefly attached to the 70S ribosome until the entire complex is dissociated through the hydrolysis of GTP in response to the binding of EF-G and another factor, called the ribosome release factor (RRF).

30.4 Eukaryotic Protein Synthesis Differs from Bacterial Protein Synthesis Primarily in Translation Initiation

The basic plan of protein synthesis in eukaryotes and archaea is similar to that in bacteria. The major structural and mechanistic themes recur in all domains of life. However, eukaryotic protein synthesis entails more protein components than does bacterial protein synthesis, and some steps are more intricate. Some noteworthy similarities and differences are as follows:

1. Ribosomes. Eukaryotic ribosomes are larger. They consist of a 60S large subunit and a 40S small subunit, which come together to form an 80S particle having a mass of 4200 kDa, compared with 2700 kDa for the bacterial 70S ribosome. The 40S subunit contains an 18S RNA that is homologous to the bacterial 16S RNA. The 60S subunit contains three RNAs: the 5S RNA, which is homologous to the bacterial 5S rRNA; the 28S RNA, which is homologous to the bacterial 23S molecules; and the 5.8S RNA, which is homologous to the 5 9 end of the 23S RNA of bacteria.

2 . Initiator tRNA. In eukaryotes, the initiating amino acid is methionine rather than N -formylmethionine. However, as in bacteria, a special tRNA participates in initiation. This aminoacyl-tRNA is called Met-tRNA i (the subscript “i” stands for initiation).

3. Initiation. The initiating codon in eukaryotes is always AUG. Eukaryotes, in contrast with bacteria, do not have a specific purine-rich sequence on the 5 9 side to distinguish initiator AUGs from internal ones.

UAA UAAUAA

RF1

Peptidecleaved

from tRNAAPE

AP

E

FIGURE 30.26 Termination of protein synthesis. A release factor recognizes a stop codon in the A site and stimulates the release of the completed protein from the tRNA in the P site.

O

O

O

adenine

OH

O

O

O

O

tRNA

O

NH

R

H

polypeptide

adenine

OH

O

O

O

O

tRNA

O

NH

R

H

polypeptide

HO

HO

H O2

+

Stryer, 2015

Liberación del polipépSdo

Péptido escindido del tRNA

Factor de liberación

Costo de la síntesis: 2 mol ATP + 2 mol GTP/mol de aminoácidos

GTP GDP + Pi

Costo de síntesis equivalente a 4 mol ATP /mol de aminoácidos

Mol aminoácidos ≈ 110 g ATP à ADP + Pi ; ∆G’ = -‐30,5 kJ (7,3 kcal) /mol

Costo de síntesis de 1 kg proteína (23 MJ) ≈ 1,11 MJ

1,11 MJ X 3= 3,3 MJ CARBOHIDRATOS

30,5 kJ/mol ATP X 4 mol ATP= 122 KJ 122 KJ/110 g AA * 1000 g AA= 1110 Kj

Eficiencia de captura de la Energía de carbohidratos en ATP: 38% ≈ 1/3


Algunas proteínas se degradan apenas minutos después de sinteSzarse: Senen una vida media corta, como la orniSna descarboxilasa (vida media: 11 minutos). Otras pueden degradarse días después de sinteSzarse, como la albúmina (vida media: 5-‐6 días) y otras meses después (hemoglobina, vida media: 120 días)

PROTEÍNAS

Síntesis

Degradación

AMINOÁCIDOS

La degradación proteica, como la síntesis, es un proceso conSnuo.

En parte, las proteinas se degradan dentro de los lisosomas, organelas que se forman en las membranas del Golgi y que degradan o reciclan componentes celulares (organelas) desgastados o materiales del medio extracelular introducidos a las células por endocitosis.

330

Protein glycosylation takes place in the lumen of the endoplasmic reticulum and in the Golgi complex

The major pathway for protein glycosylation takes place inside the lumen of the endoplasmic reticulum (ER) and in the Golgi complex, organelles that play central roles in protein trafficking (Figure 11.24). The protein is synthesized by ribosomes attached to the cytoplasmic face of the ER membrane, and the peptide chain is inserted into the lumen of the ER (Section 30.6). The N -linked glycosylation begins in the ER and continues in the Golgi complex, whereas the O -linked glycosylation takes place exclusively in the Golgi complex.

A large oligosaccharide destined for attachment to the asparagine residue of a protein is assembled on dolichol phosphate, a specialized lipid molecule located in the ER membrane and containing about 20 isoprene (C 5 ) units.

H3C

H3C H3C

CH

OP O

O OCH3

2–

n = 15–19

n

Dolichol phosphate

The terminal phosphate group of the dolichol phosphate is the site of attach-ment of the oligosaccharide. This activated (energy-rich) form of the oligosac-charide is subsequently transferred to a specific asparagine residue of the growing polypeptide chain by an enzyme located on the lumenal side of the ER.

Proteins in the lumen of the ER and in the ER membrane are trans-ported to the Golgi complex, which is a stack of flattened membranous sacs. Carbohydrate units of glycoproteins are altered and elaborated in the Golgi complex. The O -linked sugar units are fashioned there, and the N -linked sugars, arriving from the ER as a component of a glycoprotein, are modified in many different ways. The Golgi complex is the major sorting center of the cell. Proteins proceed from the Golgi complex to lysosomes, secretory gran-ules, or the plasma membrane, according to signals encoded within their amino acid sequences and three-dimensional structures (Figure 11.25).

H

H3C

H2C

CH2

Isoprene

FIGURE 11.24 Golgi complex and endoplasmic reticulum. The electron micrograph shows the Golgi complex and adjacent endoplasmic reticulum. The black dots on the cytoplasmic surface of the ER membrane are ribosomes. [Micrograph courtesy of Lynne Mercer.]

Endoplasmic reticulum

GolgiGolgi

FIGURE 11.25 Golgi complex as sorting center. The Golgi complex is the sorting center in the targeting of proteins to lysosomes, secretory vesicles, and the plasma membrane. The cis face of the Golgi complex receives vesicles from the endoplasmic reticulum, and the trans face sends a different set of vesicles to target sites. Vesicles also transfer proteins from one compartment of the Golgi complex to another. [Courtesy of Dr. Marilyn Farquhar.]

Pre-lysosome

Endoplasmicreticulum

Cis Golgi

Trans

Secretorygranule

Protein insertedin plasma membrane

Los lisosomas coneenen diferentes enzimas para degradar glúcidos, lípidos y proteínas.

712 CAPÍTULO 18 METABOLISMO LIPÍDICO I: ÁCIDOS GRASOS, TRIACILGLICEROLES Y LIPOPROTEÍNAS

Los enfoques terapéuticos actuales para reducir las concentraciones de co-lesterol se centran en la creación de inhibidores de la HMG-CoA reductasa, par-tiendo del supuesto de que deprimirán la biosíntesis de novo de colesterol y, portanto, las concentraciones intracelulares de colesterol; consecuentemente au-mentará la producción de receptores de LDL para la eliminación del colesterolextracelular de la sangre. Un inhibidor de este tipo, denominado lovastatina, fueaprobado en 1987 para el tratamiento de los pacientes con concentraciones decolesterol muy elevadas. En la actualidad, se dispone de varios otros inhibido-res de la HMG-CoA reductasa.

Actualmente, sabemos que la endocitosis mediada por el receptor es una rutamuy utilizada para la internalización de sustancias extracelulares, como otras li-

FIGURA 18.9Estructura de un hoyo revestido. Sevisualiza un hoyo revestido sobre la superficieinterna de la membrana plasmática de unacélula de mamífero en cultivo mediantemicroscopia electrónica de fractura porcongelación. La estructura de jaula del hoyo sedebe a la estructura de la clatrina, la principalproteína de estos hoyos.

Fotografía microscópica realizada por John Heuser, WashingtonUniversity School of Medicine.

FIGURA 18.10Implicación de los receptores de LDL en la captación y metabolismodel colesterol. Los receptores de LDL se sintetizan en el retículoendoplásmico y maduran en el complejo de Golgi. A continuación, migran a lasuperficie celular, en donde se agrupan en hoyos revestidos de clatrina. La LDL,formada por ésteres de colesterol y apoproteína, se une a los receptores de LDLy se internaliza en vesículas endocitósicas. Varias de estas vesículas se fusionanpara formar un orgánulo denominado endosoma. El bombeo de protones en lamembrana del endosoma hace que disminuya el pH, lo cual provoca a su vezque la LDL se disocie de los receptores. El endosoma se fusiona con un lisosoma,y la cubierta de clatrina portadora del receptor se disocia y vuelve a lamembrana. El complejo receptor-LDL se degrada en los lisosomas, y el colesteroltiene diversos destinos. Las acciones reguladoras del colesterol se indican en rojo.

EXTERIOR DE LA CÉLULA INTERIOR DE LA CÉLULA

Membranaplasmática

Receptoresde LDL

LDL Ésteresde colesterol

Apoproteína

Hoyos revestidos Vesículaendocitósica

Vesículareciclada

Endosoma

Colesterol

Retículoendoplásmico

complejode Golgi

Síntesis dereceptores de LDL(disminuida)

Gota de ésteresde colesterol

Efectosreguladoresdel colesterol

HMG-CoAreductasa

La vesícula resultante se rompe y liberala proteína LDL(morado) ycolesterol(naranja)

El endosomase fusiona conel lisosoma

Se regenera la vesículaendocitósica y vuelvea la membrana plasmática

ACAT

Aminoácidos

Lisosoma

Vesículareciclada

Cap_18.qxd 17/3/10 12:42 Página 712

Ejemplo: Captación de la LDL por endocitosis en tejidos periféricos y degradación de la apolipoproteína

lys

lys

¿Cómo reconoce la célula qué proteínas degradar?

683

pathway. In addition, cells must eliminate damaged proteins. A significant proportion of newly synthesized protein molecules are defective because of errors in translation or misfolding. Even proteins that are normal when first synthesized may undergo oxidative damage or be altered in other ways with the passage of time. These proteins must be removed before they accumu-late and aggregate. Indeed, a number of pathological conditions, such as certain forms of Parkinson disease and Huntington disease, are associated with protein aggregation.

The half-lives of proteins range over several orders of magnitude. Ornithine decarboxylase, at approximately 11 minutes, has one of the shortest half-lives of any mammalian protein. This enzyme participates in the synthesis of poly-amines, which are cellular cations essential for growth and differentiation. The life of hemoglobin, however, is limited only by the life of the red blood cell, and the lens protein crystallin is limited by the life of the organism.

23.2 Protein Turnover Is Tightly Regulated

How can a cell distinguish proteins that should be degraded? Ubiquitin (Ub), a small (76 amino acids) protein present in all eukaryotic cells, is a tag that marks proteins for destruction (Figure 23.2). Ubiquitin is the cellular equivalent of the “black spot” of Robert Louis Stevenson’s Treasure Island —the signal for death.

Ubiquitin tags proteins for destruction

Ubiquitin is highly conserved in eukaryotes: yeast and human ubiquitin differ at only 3 of 76 residues. The carboxyl-terminal glycine residue of ubiquitin becomes covalently attached to the ́ -amino groups of several lysine residues on a protein destined to be degraded. The energy for the formation of these isopeptide bonds ( iso because ́ - rather than a -amino groups are targeted) comes from ATP hydrolysis.

Three enzymes participate in the attachment of ubiquitin to a protein (Figure 23.3): ubiquitin-activating enzyme, or E1; ubiquitin-conjugating enzyme, or E2; and ubiquitin–protein ligase, or E3. First, the C-terminal carboxylate group of ubiquitin becomes linked to a sulfhydryl group of E1 by a thioester bond. This ATP-driven reaction is reminiscent of fatty acid activation (Section 22.2). In this reaction, an acyl adenylate is formed at the

NH

HN

O

OO

HN

Ub

O

H

Lys

Peptidebond

Isopeptidebond

Peptidebond

INTESTINAL CELLLUMEN BLOOD

Proteins

Amino acids

Oligopeptides

Proteolyticenzymes

Aminoacids

TripeptidesDipeptides

Peptidase

PeptidasesNa+

FIGURE 23.1 Digestion and absorption of proteins. Protein digestion is primarily a result of the activity of enzymes secreted by the pancreas. Aminopeptidases associated with the intestinal epithelium further digest proteins. The amino acids and di- and tripeptides are absorbed into the intestinal cells by specific transporters (green and orange ovals). Free amino acids are then released into the blood by transporters (red oval) for use by other tissues.

C terminus

Ubiquitin

Lys 48

FIGURE 23.2 Structure of ubiquitin. Notice that ubiquitin has an extended carboxyl terminus, which is activated and linked to proteins targeted for destruction. Lysine residues, including lysine 48, the major site for linking additional ubiquitin molecules, are shown as ball-and-stick models. [Drawn from 1UBI.pdb.]

UbiquiSna 76 AA

Extremo -‐COO-‐ terminal

En cada lisina de la proteína a degradar, se une una cadena de 4 o más Ubiquienas. Cada Ubiquiena se une por su extremo C terminal a la adyacente en el residuo de lysina 48.

UU U

U

UU U

Ulys

lys

La ubiquiSna, una proteína de 76 AA, se une por su extremo C terminal a grupos –NH2 del extremo de la cadena de carbonos de varios residuos de lisina (lys) de las proteínas marcando a las proteínas que eenen que degradarse.

U

Este eequetado de las proteínas se realiza con gasto de ATP.

Pero ¿Cómo se degradan las proteínas que están libres en el citosol?

U

U

U

U U

U UU

lys lys

ATP AMP+2Pi

E1 E2 E3 lys lys

U U

Ulys lys

ATP

AMP+2Pi E1 E2 E3

ESquetado de proteínas para degradación con UbiquiSna

U ≥ 6 ≥6 ATP ≥6 AMP+ 12 Pi

lys lys

UU U

U

UU

UU

U

E1: Enzima acevadora de Ubiquiena; E2: Enzima conjugadora de Ubiquiena; E3: Ubiquiena-‐proteína ligasa.

COSTO ≥ 8 ATP / mol proteína

UU U

U

UU U

Ulys

lys

U U

U

U

U

U

U

UAminoácidos

PépSdos

UbiquiSna

685

hundreds of members. Indeed, the E3 family is one of the largest gene fami-lies in human beings. The diversity of target proteins that must be tagged for destruction requires a large number of E3 proteins as readers.

Three examples demonstrate the importance of E3 proteins to nor-mal cell function. Proteins that are not broken down owing to a defec-

tive E3 may accumulate to create a disease of protein aggregation such as juvenile or early-onset Parkinson disease. A defect in another member of the E3 family causes Angelman syndrome, a severe neurological disorder characterized by mental retardation, absence of speech, uncoordinated movement, and hyperactivity. Conversely, uncontrolled protein turnover also can create dangerous pathological conditions. For example, human papillomavirus (HPV) encodes a protein that activates a specific E3 enzyme. The enzyme ubiquitinates the tumor suppressor p53 and other proteins that control DNA repair, which are then destroyed. The activation of this E3 enzyme is observed in more than 90% of cervical carcinomas. Thus, the inappropriate marking of key regulatory proteins for destruction can trigger further events, leading to tumor formation.

It is important to note that the role of ubiquitin is much broader than merely marking proteins for destruction. Although we have focused on pro-tein degradation, monoubiquitination also regulates proteins involved in DNA repair, chromatin remodel-ing, and protein kinase activation, among other bio-chemical processes.

The proteasome digests the ubiquitin-tagged proteins

If ubiquitin is the mark of death, what is the execu-tioner? A large protease complex called the proteasome or the 26S proteasome digests the ubiquitinated proteins . This ATP-driven multisubunit protease spares ubiqui-tin, which is then recycled. The 26S proteasome is a complex of two components: a 20S catalytic unit and a 19S regulatory unit.

The 20S unit is composed of 28 subunits, encoded by 14 genes, arranged into 4 heteroheptomeric rings stacked to form a structure resembling a barrel (Figure 23.5). The outer two rings of the barrel are made up of a -type subunits and the inner two rings of b -type subunits. The 20S catalytic core is a sealed barrel. Access to its interior is controlled by a 19S regulatory unit, itself a 700-kDa complex made up of 19 subunits. Two such 19S complexes bind to the 20S proteasome core, one at each end, to form the complete 26S proteasome (Figure 23.6). The 19S regulatory unit has three functions. First, components of the 19S unit are ubiquitin receptors that bind specifically to polyubiquitin chains, thereby ensuring that only ubiquitinated proteins are degraded. Second, an isopeptidase in the 19S unit cleaves off intact ubiquitin molecules from the proteins so that they can be reused. Finally, the doomed protein is unfolded and directed into the catalytic core. Key components of the 19S complex are six ATPases of a type called the AAA class ( A TPase a ssociated with vari-ous cellular a ctivities). ATP hydrolysis assists the 19S complex to unfold the substrate and induce conformational changes in the 20S catalytic core so that the substrate can be passed into the center of the complex.

The proteolytic active sites are sequestered in the interior of the barrel to protect potential substrates until they are directed into the barrel. There are three types of active sites in the b subunits, each with a different specificity,

TABLE 23.2 Dependence of the half-lives of cytoplasmic yeast proteins on the identity of their amino-terminal residues

Highly stabilizing residues(t1/2 . 20 hours)Ala Cys Gly MetPro Ser Thr Val

Intrinsically destabilizing residues (t1/2 5 2 to 30 minutes)Arg His Ile LeuLys Phe Trp Tyr

Destabilizing residues after chemical modification (t1/2 5 3 to 30 minutes)Asn Asp Gln Glu

Data from J. W. Tobias, T. E. Schrader, G. Rocap, and A. Varshavsky. Science 254(1991):1374–1377.

N-terminalthreonine

nucleophile

!-type subunits

!-type subunits

"-type subunits

"-type subunits

FIGURE 23.5 20S proteasome. The 20S proteasome comprises 28 homologous subunits (a-type, red; b-type, blue), arranged in four rings of 7 subunits each. Some of the b-type subunits (right) include protease active sites at their amino termini. [Subunit drawn from 1RYP.pdb.]

19S regulatory unit

20S catalytic core

19S regulatory unit

FIGURE 23.6 26S proteasome. A 19S regulatory subunit is attached to each end of the 20S catalytic unit. [From W. Baumeister, J. Walz, F. Zuhl, and E. Seemuller. Cell92(1998):367–380; courtesy of Dr. Wolfgang Baumeister.]

Proteasa (proteasoma)

ATP

Proteína eSquetada con ubiquiSna

ADP + Pi

1. Desplegar la proteína a degradar 2. Cambiar la conformación de las unidades del proteasoma para exponer la proteína a los sieos catalíecos internos. 3. Pero ¿Cuántos ATP? No sé con seguridad

pepSdasas

COSTO POR MOL DE AMINOÁCIDOS LIBERADO: INCIERTO

a eSquetado

Documents

CO2 TAG’ SÍNTESIS’Y’DEGRADACION’ … · The fidelity of protein synthesis requires accurate recognition of three-base codons on messenger RNA. Recall that the genetic code