Enzymes II Cellular Regulation of Enzymes

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Enzymes II Cellular Regulation of Enzymes

• Allosteric Enzymes
• Covalent regulation
• Proteolytic Activation
• Regulation by Isoenzymes
• Site-Directed Mutagenesis and Catalytic
  Antibodies
• Catalytic RNA

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Cellular Regulation of Enzymes Reactions
involved in a metabolic pathway are often grouped
in sequences. The first enzyme in such a pathway
is usually a regulatory enzyme that controls the
rate for the entire sequence. While the behavior
of most of the enzymes can be explained by
Michaelis-Menten kinetics, there is typically one
step that is not.
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• A B C D
  F P
• The first enzyme can be influenced by the
  concentration of the starting material A.
• It may also be affected by the amount of final
  product P.
• P inhibits E1 in a negative feedback manner.

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• End product inhibition negative feed back
  control
• Enzyme - substrate reaction is in an equilibrium
• If product builds up, the reaction slows.
• E S ES ES EP E P

Equilibrium shifts to left if product starts to
build up
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• Factors that can influence a regulatory enzyme

• concentration of the final product(s)
• beginning substrate in the pathway
• intermediates formed in the pathway
• an external factor like a hormone
• a combination of the above

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Positive and Negative Effectors

• Effectors or modulators
• Small molecules that exert influence on an
  enzyme.
• They act by reversible, noncovalent binding
  to
• the enzyme, which alters the conformation of
• the active site.

• Positive effectors - stimulate an enzyme
• Negative effectors - inhibit an enzyme

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1). Allosteric interaction Most allosteric
enzymes are oligomeric proteins with two binding
sites catalytic site and regulatory site.
  The binding of an effecter to the regulatory
site causes conformational change of the protein
and influences the activity of the catalytic
site.  
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• A positive modulator or effector is similar to a
  coenzyme.

Example of positive allosterism.
Allosteric enzymes transmit messages via
conformational changes between regulatory site
and catalytic site.
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Enzyme that are regulated by allosteric
interaction do not follow Michaelis-Menten
kinetics. The reactions usually show sigmoidal
curves.
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Since M-M kinetics is not obeyed, a Km cannot be
defined as usual. The S yieling a vo of ½
Vmax is represented as S0.5
0 enzyme substrate, - enzyme substrate
positive modulator - enzyme substrate
negative modulator.
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Cooperative or homotropic model Messages are
sent from one binding site to another via
conformational change.
 
The enzyme is very sensitive to substrate
concentration changes.
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Heterotropic model The dimer has nonidentical
subunits. Catalytic subunit ? contains the
active site. Regulatory subunit ? contains the
binding site
Step 1, Effector binds to the regulatory subunit,
which sends message to catalytic subunit. Step
2, Substrate binds more or less depending on
whether the effector is or -.
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Read p186 p187 for MWC model and sequential
model.
Reaction products
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• 2). Covalent modification
• Enzymes are covalently modified on amino acid
• side chains, so that the enzymes are activated or
  
• inactivated.
• Common alterations
• Phosphorylation of hydroxyl groups in serine,
  threonine or tyrosine.
• Attachment of an adenosine monophosphate (AMP) to
  a hydroxyl group.
• Reduction of disulfide bonds.

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• For example, synthesis of glycogen is regulated
• by the attachment or removal of a P- group to
• glycogen synthase. Fig. 7.7
• Also by by the attachment or removal of a P-
• group to glycogen phosphorylase

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(less active)
(more active)
Kinase
(less active)
(more active)
phosphatase
phosphorylase 2 H2O
phosphorylase 2 Pi
Glycogen phosphorylase is activated by
phosphorylation. The reaction is reversible and
are catalyzed by different enzymes.
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Specific serine residues in each of two identical
dimers of the enzyme are phosphorylated. The
reaction is catalyzed by phosphorylase kinase.
The process can be reversed using a second
enzyme, phosphorylase phosphatase which affects
the removal of phosphate.
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P
P
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• Other examples
• Attachment of AMP to glutamine synthetase
• Reduction of cysteine disulfide bonds by AH2

active form inactive form
active form inactive form
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  3). Proteolytic Activation Some enzymes are
synthesized in an inactive form, which is later
activated at an appropriate situation.    
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• Activation by proteolytic cleavage
• Some enzymes are initially produced in an
  inactive form - zymogen.

The removal of a short piece at the N-terminus or
C-terminus will leave an active form of the
enzyme.
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Proteolytic cleavage - breakage of peptide bonds
by peptidases or proteases.
Many digestive enzymes are regulated this way.
For. Example, (Fig. 7.8). chymotrypsinogen ?
?-Chymotrypsin ? ?-chymotrypsin   The activation
process is completed by same type of enzymes or
even the active form of the same enzyme.
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Activation of Chymotrypsin
chymotrypsinogen (inactive)
trypsin
?-chymotrypsin (active)
chymotrypsin
Ser - Arg and Thr - Asn 14 15
147 148
?-chymotrypsin (active)
The three pieces are held together by inter chain
disulfide bonds.
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• 4). Regulation by Isoenzymes
• Isoenzymes or isozymes
• Enzymes that have similar but not identical amino
  acid sequences.
• All catalyze the same biochemical reaction.
• But differ in kinetics - different KM and Vmax
  values.
• Use different effectors and coenzymes.
• Cellular distribution of each form will vary.

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• Regulation by Isoenzymes
• The best known example is lactate dehydrogenase
  (LDH) which catalyzes the conversion of pyruvate
  to lactate in muscle tissue.
• lactate NAD pyruvate NADH
• LDH is a tetramer composed of two possible types
  of subunits - M and H
• These units are made by separate genes and have
  similar but different amino acid sequences.

LDH
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LDH isoenzymes

• In skeletal muscle, the predominate form is M4
  that has high affinity to pyruvate.
• In the heart - H4 - high affinity for lactate.
• In the liver, all combinations M4, M3H, M2H2,
  MH3 and H4.
• The existence of isoenzymes may allow regulation
  based on different metabolic patterns.

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Different forms of LDH show different migration
pattern in electrophoresis. Cell damage caused
by heart attack, liver diseases, or muscle
diseases can cause LDH to leak into blood
stream. The affected tissue can be indicated by
the pattern of LDH on electrophoresis.
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(Review) Enzymes II Cellular Regulation of
Enzymes

• Allosteric Enzymes The first enzyme in a pathway
  is
• often the regulatory enzyme.
• The binding of an effecter to the regulatory
  site
• causes conformational change of the protein and
• influences the activity of the catalytic site.
• Positive effectors
• Negative effectors
• Cooperative or homotropic model
• Heterotropic model (catalytic subunit and
• regulatory subunit)

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• Covalent regulation
• Phosphorylation, (serine, threonine, tyrosine)
• Attachment of AMP, (to -OH)
• Reduction of disulfide bonds.

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Phosphorylation can activate and inactivate
Enzymes.
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Attachment of cAMP to PKA causes the enzyme to
be activated R2C2 inactive form R2 C2
active form
cAMP
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Glyceraldehyde 3-phosphate dehydrogenase is
activated by reduction of disulfide bonds.
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• Proteolytic activation
• zymogen enzyme
• Regulation by isoenzymes different kinetics,
• different cellular distribution.

protease
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• In skeletal muscle, the predominate form is M4
  that
• has high affinity to pyruvate.
• In the heart - H4 - high affinity for lactate.
• Glucose ? ? pyruvate lactate

NAD NADH NADH NAD
LDH (M4)
O2
TCA cycle
LDH (H4)
Lactate pyruvate (angina prectoris)
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Cori cycle In muscle, pyruvate is fermentated to
lactate to reproduce NAD. In liver, lactate
is reverted to pyruvate Both reactions
catalized by LDH, but different isoforms.
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• Site-directed mutagenesis
• Since 1981, recombinant DNA technique has been
  used to study the structure and function
  relationship of enzymes.
• This also allows for the design of new enzymes
  and other proteins with desired properties.

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Studies have shown that catalysts for biochemical
reactions are not limited to naturally occurring
proteins. In fact, proteins including enzymes
with any amino acid sequence can be now created
through molecular cloning technique. This
approach can be used for the design of new
therapeutic agents.
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Cystic fibrosis a genetic disease, highly
viscous mucus is produced in the lungs. Enzyme,
DNase (deoxyribonuclease) was produced through
recombinant DNA technique, and has been used on
cystic fibrosis to decrease the viscosity of the
mucous secretion in the lungs.
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Catalytic antibodies

• Enzyme substrate (transition state) ? complex
• Antibody antigen (ground state) ? complex
• Linus Pauling proposed that antibodies were
  similar to enzymes but bound to a molecules in
  the ground state.
• Studies have been conducted to see if an antibody
  with enzyme-like activity could be produced by
  using transition-state analogs as antigen.
• If so, antibodies can be produced as selective
  catalyst for desired biochemical reactions.

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• An example.
• Hydrolysis of the methyl ester of
  p-nitrobenzoate.
• The goal was to produce an antibody that bind the
  molecule of the transition state which is very
  unstable.

O
O-
C
C
O
p-nitrobenzoate
H
H
transition state
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• An analog that was easily prepared was studied.
• It was directly injected into animals, which
  resulted in antibody production.
• This antibody was found to accelerate the
  reaction by 103-106 fold.
• The catalyzed reaction was found to obey
  Michaelis-Menten kinetics.s

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Future goals of catalytic antibodies 1).
Antibodies that cleave protein in a
selective manner to assist in sequence
analysis. 2). Antibodies that cleaves proteins
or carbohy- drates that related with cancer and
viruses.
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Catalytic RNA

• Since their discovery, it was believed that all
  enzymes were proteins.
• In 1981 - 1982, two research group reported
  results on catalytic RNA.
• In 1989, the Nobel Prize in Chemistry was awarded
  to Sidney Altman (Yale) and Thomas Cech
  (University of Colorado) for their discovery.
• The term ribozyme is now used for RNA enzymes.

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Self-splicing RNA introns (Altman and Cech)

• Maturation of mRNA involves excision of precursor
  RNA splicing.
• In protozoan, Tetrahymena thermophila, it was
  observed that splicing of an intron was
  autocatalytic - it cleaved itself.

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Self-splicing RNA introns
G
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The active region of ribozymes consists of only
19 - 30 nucleotides and has a hammerhead structur
e
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Ribonuclease P

• As one example of ribozyme, ribonuclease P acts
  to remove a segment of the ribonucleotide (RNA),
  producing mature tRNA.
• The enzyme consists of a small protein subunit
  and an RNA component of 377 nucleotides.
• Substrates are at least 60 inactive, precursor
  forms of tRNA.

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RNase P functions by hydrolyitc cleavage of the
phosphodiester bond. The enzyme obeys
Michaelis-Menten Kinetics and is only needed in
small amounts.
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• List of activities for catalytic RNA
• Cleavage and rejoining of oligonucleotide
  substrates.
• Cleavage of DNA phosphodiester bonds.
• Cleavage of RNA at sequence-specific sites.
• Hydrolysis of esters.
• Formation of peptide bonds between amino acids.
• New forms of catalytic RNA continue to be
  discovered.