Enzyme Enzyme BiosynthesiBiosynthesiss

Tri Rini NuringtyasTri Rini Nuringtyas

As we remember ! Most enzymes are As we remember ! Most enzymes are proteins soproteins so

Mechanism of enzyme synthesis is no Mechanism of enzyme synthesis is no different from protein synthesis in generaldifferent from protein synthesis in general

The information which determines the The information which determines the primary sequence of an enzyme is primary sequence of an enzyme is contained in the order of DNA sequencecontained in the order of DNA sequence

From gene to proteinFrom gene to protein

DNADNA

mRNA

Transcription

Sequence of a.a

Translation

Primary structure of protein

Translation Translation is the process of "reading" is the process of "reading" the codons and linking appropriate amino the codons and linking appropriate amino acids together through peptide bondsacids together through peptide bonds

Component of translation processComponent of translation process

1.mRNA consist of genetic code

2.Ribosome

3.tRNA together with a.a

4.Enzymes

Translation process consists of 3 main stagesTranslation process consists of 3 main stages

• Initiation

• Elongation

• Termination

Initiation Activation of amino acids for incorporation intoproteins.

Activation of amino acids for incorporation into proteins.

Codon Codon sequence of three sequence of three nucleotides in a mRNA that specifies nucleotides in a mRNA that specifies the incorporation of a specific amino the incorporation of a specific amino acid into a protein.acid into a protein.

The relationship between codons and The relationship between codons and the amino acids they code for is the amino acids they code for is

called the called the genetic codegenetic code..

Genetic code Genetic code Three nucleotides - codon - Three nucleotides - codon - code for one amino acid in a proteincode for one amino acid in a protein

Not all codons are used with equal frequency.

There is a considerable amount of variationin the patterns of codon usage between different organisms.

Wobble HypothesisWobble Hypothesis

Relationships of DNA to mRNA Relationships of DNA to mRNA to polypeptide chain.to polypeptide chain.

Translation is Translation is accomplished by the accomplished by the anticodon loop of anticodon loop of tRNA forming base tRNA forming base pairs with the codon pairs with the codon of mRNA in of mRNA in ribosomesribosomes

Transfer RNA (tRNA)Transfer RNA (tRNA)composed of a nucleic acid and a specific amino acid

provide the link between the nucleic acid sequence of mRNA and the amino acid sequence it codes for.

An anticodon a sequence of 3 nucleotides in a tRNA that is complementary to a codon of mRNA

Structure of tRNAs

Two initiation factors (IF1 &IF3) bind to a 70S ribosome.promote the dissociation of 70S ribosomes into free 30S and 50S subunits.

mRNA and IF2, which carries - GTP - the charged tRNA

bind to a free 30S subunit. After these have all bound, the 30S initiation complex is complete.

Only tRNAfMet is accepted to form the initiation complex.

All further charged tRNAs require fully assembled (i.e., 70S) ribosomes

The Shine-Dalgarno sequence help ribosomes and mRNA aligns correctly for the start of translation.

Ribosome consists of- A site aminoacyl- P site peptidyl- E site exit

Peptide bond formation catalyzed by an enzyme complex called peptidyltransferase

Peptidyltransferase consists of some ribosomal proteins and the ribosomal RNA acts as a ribozyme.

The processis repeated until a termination signal is reached.

Termination of translation occurs when one of the stop codons (UAA, UAG, or UGA) appears in the A site of the ribosome.

No tRNAs correspond to those sequences, so no tRNAis bound during termination.

Proteins called release factors participate in termination

Posttranslational Processing of Posttranslational Processing of ProteinsProteins

FoldingFolding Amino acid modification (some proteins)Amino acid modification (some proteins) Proteolytic cleavageProteolytic cleavage

FOLDING Before a newly translated polypeptide can be active, it must be folded into the proper 3-D structure and it may have to associate with other subunits.

Enzymes/protein involve in folding process

1. Cis-trans isomerase for proline

Proline is the only amino acid in proteins forms peptide bonds

in which the trans isomer is only slightly favored (4 to 1 versus

1000 to 1 for other residues).

Thus, during folding, there is a significant chance that the wrong proline isomer will form first. Cells have enzymes to catalyze the cis-trans isomerization necessary to speed correct folding.

2. disulfide bond making enzymes 3. Chaperonins (molecular chaperones) a protein to help keep it properly folded and non-

aggregated.

Insulin is synthesized single polypeptide preproinsulin

has leader sequence(help it be transported through the cell membrane)

Specific protease cleaves leader sequence proinsulin.

Proinsulin folds into specific 3D structure and disulfide bonds form

Another protease cuts molecule insulin 2 polypeptide chains

Chaperones Function to keep a newly synthesized protein from either improperly folding or aggregating

After synthesized, protein needs to fold in order to have its function

The folding pattern is dictated in the amino acid sequence of the protein.

a. Some proteins capable to fold into its proper 3-D structure by itself without any help of other molecules

b. Some proteins need chaperones to fold (example in human hsp 70)

c. Some proteins need bigger protein chaperonins to be able to fold correctly.

Chaperonins a polysubunit protein form “a cage” like shape give micro environment to protein

Protein Protein TargetingTargeting

Nascent proteins contain signal sequence determine their ultimate destination.

Bacteria newly synthesized protein can: stay in the cytosol, send to the plasma membran, outer membrane, periplasmic, extracellular.

Eukaryotes can direct proteins to internal sites lysosomes, mitochondria etc.

Nascent polypeptide E.R and glycosylated golgi complex and modified sorted for delivery to lysosomes, secretory vesicle and plasma membrane.

TranslocationTranslocation The protein to be translocated The protein to be translocated

(called (called a pro-proteina pro-protein) is ) is complexed in the cytoplasm complexed in the cytoplasm with a chaperone with a chaperone

The complex keeps the The complex keeps the protein from folding protein from folding prematurely, which would prematurely, which would prevent it from passing prevent it from passing through the secretory porean through the secretory porean ATPase that helps drive the ATPase that helps drive the translocationtranslocation

after the pro-protein is after the pro-protein is translocated, the leader translocated, the leader peptide is cleaved by a peptide is cleaved by a membrane-bound protease membrane-bound protease and the protein can fold into and the protein can fold into its active 3-d form.its active 3-d form.

Signal recognition particle (SRP) detects signal sequence and brings ribosome to the ER membrane

Most Most mitochondrial mitochondrial proteins are proteins are synthesized in synthesized in the cytosol and the cytosol and imported into imported into the organellethe organelle

Control of enzyme biosynthesisControl of enzyme biosynthesis

In living cell In living cell not all enzymes not all enzymes are synthesized with maximum are synthesized with maximum velocity all the time.velocity all the time.

The rate of enzymes production The rate of enzymes production controlled in accordance w/ controlled in accordance w/

• metabolic need

• state of development of the cell

The main point in the control of enzyme synthesis

copying of the genes of the DNA in the form of

mRNA

The inhibition of enzyme synthesis The inhibition of enzyme synthesis known as known as repressionrepression

The operon model, as proposed in 1961 by Jacob and Monod.

A A B B

The rates of formation of enzyme which are The rates of formation of enzyme which are controlled by repressor controlled by repressor regulated by the regulated by the metabolic state of the cellmetabolic state of the cell

[A] [A] too high too high induction by substrateinduction by substrate

[B] [B] too high too high repression by productrepression by product

Melibiose and IPTG a good inducer but not the substrate of galactose

Gal repressor the action is prevented by the presence of D-galactose

Repressor does not by itself bind to the operator has specific binding site for the product

So repressor-product bind to the operator

Example amino acid synthesis

Enzyme turn overEnzyme turn over

Proteins are targeted for destruction

Proteins have different half-livesMost enzymes that are important in metabolic regulation have short lives

Also important for removal of abnormal proteins / enzymes

Proteolytic enzymes are found through out the cell

ubiquitinA small protein present in all eukaryotic cells

tagging protein for destruction

Several proteases present in the eukaryotic cytosoltwo Ca2+ activated proteases calpains

an ATP-dependent protease proteasome

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

1. Ubiquitination

2. Oxidation of amino acid residues

3. PEST sequences

4. N-terminal amino acid residue

Three enzymes participate in the conjugation of ubiquitin to proteins1. Terminal carboxyl of

ubiquitin link to a sulfhydril group of E1

2. Activated ubiquitin then shuttled to a sulfhydril of E2

3. Target protein is tagged by ubiquitin for degradation

4. Ubiquitin-specific protease recognize the target degrade

2. Oxidation of amino acid residuesConditions that generate oxygen radicals cause

many proteins to undergo mixed-function oxidation of particular residues

Conditions require Fe2+ and hydroxyl radical, and the amino acids most susceptible to oxidation are

lysine, arginine, and proline.E. coli and rat liver contain protease cleaves

oxidized glutamine synthetase in vitro, but does not attack the native enzyme

accumulation of oxidatively damaged protein beyond the cell’s capacity to degrade and replace them contribute to importantly to cellular aging

3. PEST sequence3. PEST sequenceall short-lived proteins (i.e., half-lives < 2 h) all short-lived proteins (i.e., half-lives < 2 h)

contain 1 or more regions rich in contain 1 or more regions rich in

proline, glutamate, serine, and threonineproline, glutamate, serine, and threonine

4. N-terminal amino acid residue4. N-terminal amino acid residue

An N-terminal protein residue of An N-terminal protein residue of Phe, Leu, Tyr, Trp, Lys, or ArgPhe, Leu, Tyr, Trp, Lys, or Arg short metabolic short metabolic

lifetimeslifetimes

. Proteins with other termini are far longer-lived. . Proteins with other termini are far longer-lived. Thus, the intracellular half-life of a particular protein Thus, the intracellular half-life of a particular protein depends on the identity of its N-terminal amino acid depends on the identity of its N-terminal amino acid residue.residue.

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