Regulatory Strategies
Enzymes and Hemoglobin
Regulatory Strategies
What are the four ways in which enzymes
are regulated?
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allosteric regulation
enzymes exist in multiple forms (isoenzymes)
covalent modification
proteolytic cleavage
Allosteric Regulation
 What are some of the characteristics of this form
of regulation?
– Activity influenced by non-covalent binding of
metabolite called a modulator
• May be inhibitory or stimulatory
• May have one (monovalent) or several (polyvalent)
modulators
• Binding induces shape change in enzyme
• Enzymes are large; two or more subunits
• Exhibit homotropic or heterotropic control
Allosteric Regulation
– enzyme may be part of a sequence in which
the end product inhibits allosteric enzyme
– enzyme does not show Michaelis-Menten
kinetics
Aspartate Transcarbamoylase
 What is the reaction catalyzed by this enzyme?
Aspartate Transcarbamoylase
 CTP is a negative modulator and ATP is a
positive modulator
– feedback inhibition
Aspartate Transcarbamoylase
 How do we know that
the catalytic and
regulatory sites on this
enzyme are distinct?
– treat with
p-hydroxymercuribenzoate
Aspartate Transcarbamoylase
 Catalytic subunit consists of 3 chains (c3) and
regulatory subunit consists of 2 chains (r2)
Aspartate Transcarbamoylase
 Allosteric modulators alter the quaternary
structure of the enzyme
Aspartate Transcarbamoylase
What is the difference between the T state
and the R state of the enzyme?
– T = tense; lower affinity for substrate
– R = relaxed; higher affinity for substrate
Binding of substrate or substrate analog
converts enzyme from T to R state
– positive cooperativity
– homotropic control
Aspartate Transcarbamoylase
 How does CTP act as
a negative modulator?
 Why is this an
example of
heterotropic
modulation?
Aspartate Transcarbamoylase
How does ATP act as a positive modulator?
Does this represent homo or heterotropic
modulation?
Aspartate Transcarbamoylase
 Why is the
mechanism just
described called the
concerted model?
– All subunits must be
in same conformation,
T or R
Allosteric Regulation
 An alternative, the sequential model has been
proposed
Allosteric Regulation
 How does the sequential model differ from the
concerted model?
– Subunits may undergo individual sequential changes
in conformation
– Subunits can interact even in different conformations
– Change induced by binding of substrate to one
subunits can increase or decrease substrate binding to
other subunits
• positive or negative homotropic effects
• finer tuning
Allostery and Hemoglobin
 Binding of O2 to
hemoglobin
represented by a
sigmoidal curve
similar to allosteric
enzymes
 Cooperativity
promotes efficient O2
delivery
Allostery and Hemoglobin
 What part of the hemoglobin molecule binds O2?
Allostery and Hemoglobin
 How does the binding of O2 affect the structure of
heme?
Allostery and Hemoglobin
 How does O2 binding influence the quaternary
structure of hemoglobin?
Allostery and Hemoglobin
 How does 2,3 bisphosphoglycerate affect O2
affinity of hemoglobin?
 How is this effect brought about?
Allostery and Hemoglobin
 How is fetal
hemoglobin different
from maternal
hemoglobin?
 How can this be
explained?
Allostery and Hemoglobin
 What is the Bohr effect?
 What is the chemical basis of this effect?
Allostery and Hemoglobin
 CO2 also stabilizes deoxyhemoglobin by forming
carbamate groups
Isozymes
What are isozymes or isoenzymes?
– enzymes that have differences in amino acid
sequence but catalyze the same reaction
• Example – lactate dehydrogenase
• Two different chains, M and H, exist
• Enzyme consists of 4 subunits
– H4, H3M, H2M2, HM3, M4
• Each form has different Km and Vmax
Isozymes
 M4 functions best in anaerobic environment
while H4 in aerobic environment
Covalent Modification
Enzymes exist in active and inactive forms
– Interconvertable by covalent modification
• Catalyzed by other enzymes
• Most modifications are reversible
Covalent Modification
Covalent Modification
What are the most common forms of
covalent modification?
– Phosphorylation and dephosphorylation
Which enzymes catalyze phosphorylation?
– protein kinases
Which enzymes catalyze
dephosphorylation?
– protein phosphatases
Covalent Modification
 What donates the phosphate group?
Covalent Modification
Why is phosphorylation an effective way
to regulate proteins?
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–
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Phosphate group adds negative charges
Phosphate group can form hydrogen bonds
Free energy of phosphorylation is large
Can occur rapidly or slowly as needed
Can achieve amplification
Linked to energy status of cell
Covalent Modification
Example – glycogen phosphorylase
– Two forms a and b
• A = active; 4 subunits each with a serine residue
phosphorylated at OH group
• B = inactive; removal of PO4 groups causes protein
to separate into two half molecules
Covalent Modification
 What is the connection between c-AMP and
protein kinases?
– PKA activated by c-AMP in cells
Zymogens
What are zymogens?
– inactive precursors of enzymes
How are they activated?
– proteolytic cleavage
Zymogens
Zymogens
What are some other examples?
– Blood clotting
– Insulin
– Conversion of procollagenase to collaginase in
metamorphosis
– Conversion of procaspases to caspases in
apoptosis
Zymogens
 Activation of
chymotrypsinogen to
chymotrypsin
Zymogens
 Why is trypsin a key enzyme in zymogen
activation?
Zymogens
Why do proteolytic enzymes have specific
inhibitors?
– To prevent premature activation of the enzyme
– Activation of trypsin in pancreas could destroy
pancreatic tissue
Zymogens
 How are zymogens
involved in the
formation of blood
clots?