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Enzyme Biosynthesis. Tri Rini Nuringtyas. As we remember ! Most enzymes are proteins so Mechanism of enzyme synthesis is no different from protein synthesis in general The information which determines the primary sequence of an enzyme is contained in the order of DNA sequence. - PowerPoint PPT Presentation
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Enzyme Enzyme BiosynthesiBiosynthesiss
Tri Rini NuringtyasTri Rini Nuringtyas
As we remember ! Most enzymes are proteins As we remember ! Most enzymes are proteins soso
Mechanism of enzyme synthesis is no different Mechanism of enzyme synthesis is no different
from protein synthesis in generalfrom protein synthesis in general
The information which determines the The information which determines the primary sequence of an enzyme is contained primary sequence of an enzyme is contained in the order of DNA sequencein the order of DNA sequence
From gene to proteinFrom gene to protein DNADNA
mRNATranscription
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 code2.Ribosome3.tRNA together with a.a4.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 biosynthesisIn 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 BThe rates of formation of enzyme which are controlled The rates of formation of enzyme which are controlled
by repressor by repressor regulated by the metabolic state of regulated by the metabolic state of the cellthe 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 productSo repressor-product bind to the operatorExample amino acid synthesis
Enzyme turn overEnzyme turn overProteins are targeted for destructionProteins have different half-livesMost enzymes that are
important in metabolic regulation have short livesAlso 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 residues3. 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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