Download Enzyme Biosinthess

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
From gene to protein
DNA
Transcription
mRNA
Translation
Sequence
of a.a
Primary structure of
protein

Translation  is the process of "reading" the
codons and linking appropriate amino acids
together through peptide bonds

Component 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 stages
• Initiation
• Elongation
• Termination
Initiation
Activation of amino acids for
incorporation into
proteins.
Activation of amino acids for
incorporation into proteins.
Genetic code  Three nucleotides - codon - code
for one amino acid in a protein
Codon  sequence of three nucleotides in a
mRNA that specifies the incorporation of a
specific amino acid into a protein.
The relationship between codons and the
amino acids they code for is called the
genetic code.
Not all codons
are used with
equal
frequency.
There is a
considerable
amount of
variation
in the patterns of
codon usage
between different
organisms.
Wobble Hypothesis
Relationships of DNA to mRNA to
polypeptide chain.
Translation is
accomplished by the
anticodon loop of tRNA
forming base pairs
with the codon of
mRNA in ribosomes
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.
Structure of tRNAs
An anticodon  a
sequence of 3 nucleotides
in a tRNA that is
complementary to a
codon of mRNA
Only tRNAfMet is accepted to
form
Twothe
initiation
initiation
factors
complex.
(IF1
&IF3) bind to a 70S
Allribosome.
further charged tRNAs
require
promote
fully assembled
the dissociation
(i.e.,
70S)
of 70S
ribosomes
ribosomes into free
30S and 50S subunits.
The Shine-Dalgarno
sequence
help
ribosomes
mRNA andIF2,
which
and
carries
mRNA aligns correctly for
the
- GTP
start of translation.
- the charged tRNA
Ribosome consists of
- bind
A site 
toaminoacyl
a free 30S subunit.
-
P site
After
these
peptidyl
have all
- bound,
E site the
exit30S initiation
complex is complete.
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 process
is 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 tRNA
is bound during termination.
Proteins called release factors
participate in termination
Posttranslational Processing of Proteins
Folding
 Amino acid modification (some proteins)
 Proteolytic 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 
a. Some proteins capable to
Function
to its
keep
a newly
fold into
proper
3-D
synthesized
protein
structure by
itselffrom
without
either
folding
or
any improperly
help of other
molecules
aggregating
b. Some proteins need
chaperones
to fold
After
synthesized,
protein
(example
hsp
70)
needs
to foldininhuman
order to
have
its
function
c. Some
proteins need bigger
protein  chaperonins to
The
is dictated
befolding
able topattern
fold correctly.
in the amino acid sequence of
the protein.
Chaperonins  a polysubunit
protein form “a cage” like
shape  give micro
environment to protein
Protein Targeting
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.

Translocation 



The protein to be translocated
(called a pro-protein) is
complexed in the cytoplasm with
a chaperone
The complex keeps the protein
from folding prematurely, which
would prevent it from passing
through the secretory porean
ATPase that helps drive the
translocation
after the pro-protein is
translocated, the leader peptide is
cleaved by a membrane-bound
protease and the protein can fold
into its active 3-d form.
Signal recognition particle (SRP) detects signal
sequence and brings ribosome to the ER membrane
Most
mitochondrial
proteins are
synthesized in the
cytosol and
imported into the
organelle
Control of enzyme biosynthesis
In living cell  not all enzymes
are synthesized with maximum
velocity all the time.
The rate of enzymes production
 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  known as repression
The operon model, as proposed in 1961 by Jacob and Monod.
A  B
The rates of formation of enzyme which are controlled by
repressor  regulated by the metabolic state of the cell
[A]  too high  induction by substrate
[B]  too high  repression by product
Repressor
does not
itself
to the
operator
 has
Melibiose
and by
IPTG
 bind
a good
inducer
but not
the
specificsubstrate
binding site
for the product
of galactose
So repressor-product  bind to the operator
Gal repressor  the action is prevented by the
Example
 amino
acid synthesis
presence
of D-galactose
Enzyme turn over
Proteins are targeted for destruction
Proteins have different half-lives
Most 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
Several proteases  present in the eukaryotic cytosol
two 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
ubiquitin
A small protein present in all eukaryotic
cells
 tagging protein for destruction
Three enzymes participate
in the conjugation of
ubiquitin to proteins
1. 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 residues
 Conditions
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 sequence
all short-lived proteins (i.e., half-lives < 2 h) 
contain 1 or more regions rich in
proline, glutamate, serine, and threonine
4. N-terminal amino acid residue
An N-terminal protein residue of
Phe, Leu, Tyr, Trp, Lys, or Arg  short metabolic
lifetimes
. Proteins with other termini are far longer-lived. Thus,
the intracellular half-life of a particular protein depends
on the identity of its N-terminal amino acid residue.
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