Transcript Lecture 12

Enzyme Regulation - Regulatory Strategies
• Enzyme regulation by
– Allosteric control (include feedback inhibition)
– Stimulation and inhibition by control proteins
– Reversible covalent modification
– Proteolytic activation
Allosteric Control
Example: Feedback Inhibition
• Feedback inhibition
– Seen in multi-enzyme systems
– One enzyme acts as a regulatory enzyme
– When end product exceeds cell’s requirement, it
inhibits specifically the regulatory enzyme
– All other enzymes in the system are slowed as a
result of lowered substrate level.
• Example: E1 of the L-Ile biosynthesis (from LThr) enzyme system
– E1: Threonine dehydratase, is specifically
inhibited allosterically by L-Ile, the end of
product, but not by any of the four intermediates
– L-Ile binds at a regulatory not the active site
.
Activation of Protein Kinase A by cAMP (Allosteric Control)
• Protein Kinase A (PKA)
– Alters activities of target proteins by
phosphorylating specific Ser/Thr
– Activated by cAMP
– Mechanism of activation (allosteric)
• R2C2 (in the absence of cAMP)
– 2 catalytic unit, high affinity
– 2 regulatory unit, high affinity for cAMP
• R2 and 2C (in the presence of cAMP)
• R chain binds C chain
– pseudosubstrate sequence
(Arg-Arg-Gly-Ala-Ile) in R
– Blocks active site of C
• cAMP binding
– Induces conformational change in R
– Causes dissociation of C from R
(unblock active site)
Covalent Modification
• Some regulatory enzymes undergo reversible
covalent modification
– Modifying groups
• Phosphoryl (-PO32-) (on Tyr, Ser, Thr, His)
• adenylyl (Tyr)
• uridylyl (Tyr)
• adenosine diphosphate ribosyl (Arg, Gln, Cys,
diphthamide - a modified His)
• methyl groups (Glu)
– Covalently linked to and removed from the
regulatory enzyme by separate enzymes
Enzyme Regulation by Phosphorylation
• Phosphorylation
– Most common type of reversible covalent modification
– Affects structure and therefore regulates activities of many enzymes
– Phosphorylation carried out by protein kinases
R-OH + ATP
R-O-PO32- + ADP + H+
– Protein dephosphorylation carried out by protein phosphatases
R-O-PO32- + H2O
R-OH + Pi
Residues that can be phosphorylated: Ser, Thr, Tyr
Effects of Phosphylation
(Phosphoryl groups affect the structure and catalytic activity of proteins)
• Adds to negative charges  electrostatic
interaction  structural changes  change in
substrate binding or catalytic activity
(e.g., repel negative charges on Glu/Asp or
favorable interaction either electrostatic or Hbonding with Arg)
• Phosphoryl group capable of 3 H-bonds,
highly directional
• Phosphorylation is fast  enzyme can be
turned on/off fast
• Example: glycogen phosphorylase
Glycogen Phosphorylase
• Glycogen Phosphorylase a and b
differ in their secondary, tertiary,
and quaternary structures
• The active site undergoes changes
in structure and, consequently
changes in catalytic activity as a
consequence of phosphorylation
/dephosphorylation
• How? N-term 20 amino acids
(contains basic residues such as
Arg) interact with acidic residues
somewhere else. Phosphorylation
of Ser14 disrupts these interaction
and results in conformational
change.
Less active
Ser
Ser
More active
Ser phosphorylation site (Yellow)
Subunit 2
Allosteric activator AMP (dark blue)
Active site
Pyridoxal phosphate
(PLP, light blue) (Vit B6 derivative)
Glucose (red) bound at active site
Phosphorylase a
Subunit 1
Proteolytic Activation: Zymogen to Active Protease
• Activation of proteases
– Pepsinogen (stomach) to pepsin
– Trypsinogen (pancrease) to trypsin
– Chymotrypsinogen (pancrease) to chymotrypsin
– Procarboxypeptidase (pancrease) to carboxypeptidase
– Proelastase (pancrease ) to elastase
– Blood clotting enzymes
• Proinsulin (protein hormone) to insulin
• Procollagenase to collagenase
• Activation of zymogens in the control of developmental
processes
Trypsinogen is the Common Activator
of All the Pancreatic Zymogens
• Concurrent action of digestive proteases in duodenum
• Trypsin activates:
trypsinogen
chymotrypsinoge
proelastase
procarboxypeptidase
What activates
trypsinogen first to
produce trypsin?
Enteropeptidase
(secreted by duodenum)
Chymotrypsinogen Activation slide 1
• Chymotrypsinogen (inactive)
 chymotrypsin (active)
– chymotrypsinogen
(245aa single chain) to
-chymotrypsin (active)
by trypsin
– -chymotrypsin to chymotrypsin by
chymotrypsin
Mechanism of Chymotrypsinogen Activation slide 2
• Newly formed N-term
(Ile16) (+ly charged)
turns inward and interacts
with Asp194
• Induces conformational
change
• Incomplete substrate binding
site becomes complete
• Summary: Hydrolysis of a
single peptide bond results in
highly localized
conformational changes that
switches the enzymatic
activity of the enzyme
Pancreatic Trypsin Inhibitor
• Zymogen (inactive)  active enzyme
– Proteolysis (irreversible)
• Active enzyme  inactive enzyme
– Protease inhibitors
• Pancreatic trypsin inhibitor (6 kDa)
– Kd = 0.1 pM (tight binding to trypsin)
– Complex cannot be dissociated with 8 M urea
or 6 M guianidine
– A very effective substrate analog
– Almost perfectly complementary to active site
1-antitrypsin and Pulmonary Emphysema
• 1-antitrypsin (1-antiproteinase) (plasma protein)
– Protects tissue from digestion by elastase
(secreted by neutrophils, white blood cells that engulf bacteria)
– Blocks elastase much better than trypsin
• 1-antitrypsin deficiency and emphysema
– Genetic disorders lead to 1-antitrypsin deficiency
– Excess elastase digests elastic fibers (elastin & collagen
type IV) and other connective tissue proteins
– This destroys alveolar walls in the lungs  emphysema
• Cigarette smoking increases the likelihood of
develop emphysema
– Smoke oxidizes Met358 of the inhibitor, essential for
elastase binding
Pepsinogen Activation
• Pepsin (digest proteins in the highly acidic environment of the stomach)
– pH optimum 2
– Catalytic residues: 2 Asp
– First 44 amino acids removed proteolytically and
spontaneously below pH 5
• Mechanism of activation
– Highly basic precursor segment (6 Arg&Lys, +
charged)
– Highly acidic pepsin moiety (including catalytic
Asp residues)
– Fully formed active site is blocked in zymogen form
– Lowering pH below 5 disrupts salt bridges and
exposes active site