This document discusses various ways that enzyme activity can be regulated. It describes how enzyme levels and activity can be increased or decreased through different mechanisms, including substrate availability, product accumulation, allosteric effectors, covalent modification, genetic controls, zymogens, isozymes, and modulator proteins. A key example discussed is allosteric regulation, where binding of an allosteric effector at a site other than the active site can increase or decrease an enzyme's catalytic efficiency. Phosphorylation is provided as a common example of covalent regulation, where the addition of phosphate groups can alter an enzyme's activity.

1. Lecture 12 Enzyme regulation

2. What Factors Influence Enzymatic
Activity?
• Two of the more obvious ways to regulate
the amount of activity are
1. To increase or decrease the number of
enzyme molecule (enzyme level)
2. To increase or decrease the activity of each
enzyme molecule (enzyme activity)

3. • A general overview of factors influencing
enzyme activity includes the following
considerations
1. Rate depends on substrate availability
2. Rate slows as product accumulates
3. Allosteric effectors may be important
4. Enzymes can be modified covalently
5. Genetic controls (transcription regulation) -
induction and repression (enzyme level)
6. Zymogens, isozymes and modulator proteins
may play a role

4. • A general overview of factors influencing
enzyme activity includes the following
considerations
1. Rate depends on substrate availability
2. Rate slows as product accumulates
3. Allosteric effectors may be important
4. Enzymes can be modified covalently
5. Genetic controls (transcription regulation) -
induction and repression (enzyme level)
6. Zymogens, isozymes and modulator proteins
may play a role

5. Non-covalent Interactions
Substrate availability
• Non-regulatory enzymes generally exhibit
hyperbolic kinetics (Michaelis-Menton)
• At low substrate concentration, reaction rate
proportional to substrate concentration
• Regulatory enzymes generally exhibit
sigmoidal kinetics (positive cooperativity)
• Changes of substrate concentrations at normal
physiological levels greatly alter reaction rate

6. • Regulatory enzymes are usually the
enzymes that are the rate-limiting step, in
a pathway, meaning that after this step a
particular reaction pathway will go to
completion

7. • A general overview of factors influencing
enzyme activity includes the following
considerations
1. Rate depends on substrate availability
2. Rate slows as product accumulates
3. Allosteric effectors may be important
4. Enzymes can be modified covalently
5. Genetic controls (transcription regulation) -
induction and repression (enzyme level)
6. Zymogens, isozymes and modulator proteins
may play a role

8. Non-covalent Interactions
Allosteric Regulation
• Binding of allosteric effectors at allosteric
sites affect catalytic efficiency of the
enzyme

9. Non-covalent Interactions
Allosteric Regulation
• Allosteric Activators
– Decrease Km (increases the enzyme
binding affinity)
– Increases Vmax (increases the enzyme
catalytic efficiency)

10. Non-covalent Interactions
Allosteric Regulation
• Allosteric Inhibitors
– Increases Km (decreases enzyme binding
affinity)
– Decreases Vmax (decreases enzyme catalytic
efficiency)

11. Molecules that act as allosteric effectors
• End products of pathways
– Feedback inhibition
• Substrates of pathways
– Feed-forward activators
• Indicators of Energy Status
– ATP/ADP/AMP
– NAD/NADH
– Citrate & acetyl CoA

12. Allosteric Example
• Feedback Inhibition - This occurs
when an end-product of a pathway
accumulates as the metabolic demand
for it declines.
• This end-product in turn binds to the
regulatory enzyme at the start of the
pathway and decreases its activity - the
greater the end-product levels the
greater the inhibition of enzyme activity.

13. Metabolic Pathway Product/
Feedback Inhibition

14. Feed-forward activators
Phosphofructokinase ( PFK)
Fructose-6-P + ATP -----> FFrruuccttoossee--11,,66--bbiisspphhoosspphhaattee + AADDPP
•PFK catalyzes 1st committed step in glycolysis (10 steps total)
(Glucose + 2ADP + 2 NAD+ + 2Pi  2pyruvate + 2ATP + 2NADH)
•ADP is an allosteric activator of PFK

15. Allosteric modulators bind to site other than the
active site
Fructose-6-P + ATP -----> FFrruuccttoossee--11,,66--bbiisspphhoosspphhaattee + AADDPP
AADDPP
Allosteric Activator (ADP)
binds distal to active site
PFK exists as a homotetramer in bacteria and mammals

16. Vo vs [S] plots give sigmoidal curve
for at least one substrate
Activator can shift hyperbolic (as if there were no T state)
Binding of this allosteric inhibitor or this activator does not
effect Vmax, but does alter Km
Allosteric enzyme do not follow M-M kinetics

17. Sample questions
• Two curves showing the rate versus substrate concentration are
shown below for an enzyme catalyzed reaction. One ‐ curve is for the
reaction in the presence of substance X. The other curve is for data
in the absence of substance X. Examine the curves and tell which
statement below is true.
• A) The catalysis shows Michaelis‐Menten kinetics with or without X.
• B) X increases the activation energy for the catalytic reaction.
• C) X could be a competitive inhibitor.
• D) X is an activator of the enzyme.

18. Sample questions
Allosteric enzymes are
• A.similar to simple enzyme
• B.smaller than simple enzyme
• C.larger and more complex than simple enzyme
• D.smaller than simple enzyme but not complex
Which statement is false about allosteric regulation?
• A. It is usually the mode of regulation for the last step in reaction pathways
since this step produces the final product.
• B. Cellular response is faster with allosteric control than by controlling
enzyme concentration in the cell.
• C. The regulation usually is important to the conservation of energy and
materials in cells.
• D. Allosteric modulators bind non-covalently at sites other than the active
site and induce conformational changes in the enzyme.

19. Sample questions
Allosteric modulators seldom resemble the substrate or
product of the enzyme. What does this observation
show?
• A) Modulators likely bind at a site other than the active
site.
• B) Modulators always act as activators.
• C) Modulators bind non-covalently to the enzyme.
• D) The enzyme catalyzes more than one reaction.

20. Sample questions
• Some enzymatic regulation is allosteric. In such cases,
which of the following would usuallybe found?
• A) cooperativity
• B) feedback inhibition
• C) both activating and inhibitoryactivity
• D) an enzyme with more than one subunit
• E) the need for cofactors

21. Sample questions
• Describe allosteric regulation of
enzyme activity?
An allosteric enzyme is one in which the activity of the enzyme can be
controlled by the binding of a molecule to the “allosteric site”, somewhere
other than the active site. Thus allosteric control of an enzyme can be
classed in two ways. A positive allosteric regulation is the binding of a
molecule to the enzyme which increase the rate of reaction. The opposite is
a negative allosteric regulation. An example for this is phosphofructokinase,
which is promoted by a high AMP concentration, and inhibited by a high ATP
concentration.

22. Non-covalent Interactions
Protein-Protein Interactions
• Calmodulin (CALcium MODULted proteIN)
– Binding of Ca++ to calmodulin changes its
shape and allows binding and activation of
certain enzymes

23. Binding of calcium to Calmodulin
changes the shape of the protein
Unbound Calmodulin
on left
Calcium bound
Calmodulin on right.
Stars indicate exposed
non-polar ‘grooves’
that non-covalently
binds proteins

24. Calmodulin
• Extracellular [Ca] = 5 mM
• Intracellular [Ca] = 10-4 mM
– Bound Ca can be released by hormonal
action, nerve innervation, light, ….
– Released Ca binds to Calmodulin which
activates a large number of proteins

25. Calmodulin plays a role in:
• Muscle contraction
• Inflammation
• Apoptosis
• Memory
• Immune response….
• Metabolism
– Activates phosphorylase kinase
• Stimulates glycogen degradation during exercise

26. • A general overview of factors influencing
enzyme activity includes the following
considerations
1. Rate depends on substrate availability
2. Rate slows as product accumulates
3. Allosteric effectors may be important
4. Enzymes can be modified covalently
5. Genetic controls (transcription regulation) -
induction and repression (enzyme level)
6. Zymogens, isozymes and modulator proteins
may play a role

27. Covalent Regulation of Enzyme Activity
Phosphorylation and Dephosphorylation
• Addition or deletion of phosphate groups
to particular serine, threonine, or tyrosine
residues alter the enzymes activity

28. Enzymes regulated by covalent modification are called interconvertible
enzymes.
The enzymes (protein kinase and protein phosphatase) catalyzing the conversion
of the interconvertible enzyme between its two forms are called converter
enzymes. In this example, the free enzyme form is catalytically active, whereas
the phosphoryl-enzyme form represents an inactive state.
The -OH on the interconvertible enzyme represents an -OH group on a specific
amino acid side chain in the protein (for example, a particular Ser residue)
capable of accepting the phosphoryl group.

29. Covalent Regulation of Enzyme Activity
Enzyme Cascades
• Enzymes activating enzymes allows for
amplification of a small regulatory signal

30. Sample questions
• Which statement is false about covalent modification?
• A) It is reversible.
• B) It is slower than allosteric regulation.
• C) It is irreversible.
• D) Phosphorylation is a common covalent modification.

31. Sample questions
Protein kinases are enzymes that act on other enzymes by
adding phosphates groups. When the enzyme is
phosphorylated, it changes its activity (it becomes more
or less active, depending on the enzyme). This
regulatory mechanism of enzymatic activity is called:
• A) Allosteric Control
• B) Competitive inhibition
• C) Covalent Modification
• D) Isozymes Modification
• E) Zymogen activation

32. • A general overview of factors influencing
enzyme activity includes the following
considerations
1. Rate depends on substrate availability
2. Rate slows as product accumulates
3. Allosteric effectors may be important
4. Enzymes can be modified covalently
5. Genetic controls (transcription regulation) -
induction and repression (enzyme level)
6. Zymogens, isozymes and modulator proteins
may play a role

33. Changes in Enzyme Abundance
• Inducible vs Constitutive Enzymes
• Induction is caused by increases in rate of
gene transcription.
– Hormones activate transcriptional factors
• Increase synthesis of specific mRNA
• Increase synthesis of specific enzymes

34. Regulation of Enzyme
Concentrations: Induction
• Induction (an increase caused by an effecter
molecule) of enzyme synthesis is a common
mechanism - this can manifest itself at the level of
gene expression, RNA translation, and post-translational
modifications. The actions of many
hormones and/or growth factors on cells will ultimately
lead to an increase in the expression and translation
of "new" enzymes not present prior to the signal.

35. Regulation of Enzyme
Concentrations: Degradation
• The degradation of proteins is constantly
occurring in the cell.
• Proteolytic degradation is an irreversible
mechanism.

36. Regulation of Enzyme
Concentrations: Degradation (cont)
• Protein degradation by proteases is
compartmentalized in the cell in the lysosome (which
is generally non-specific), or in macromolecular
complexes termed proteasomes.
• Degradation by proteasomes is regulated by a
complex pathway involving transfer of a 76 aa
polypeptide, ubiquitin, to targeted proteins.
Ubiquination of protein targets it for degradation by
the proteasome. This pathway is highly conserved in
eukaryotes, but still poorly understood

37. • A general overview of factors influencing
enzyme activity includes the following
considerations
1. Rate depends on substrate availability
2. Rate slows as product accumulates
3. Allosteric effectors may be important
4. Enzymes can be modified covalently
5. Genetic controls (transcription regulation) -
induction and repression (enzyme level)
6. Zymogens, isozymes and modulator proteins
may play a role

38. Covalent Regulation of Enzyme Activity
Limited Proteolysis
• Specific proteolysis can activate certain
enzymes and proteins (zymogens)
– Digestive enzymes
– Blood clotting proteins
– Peptide hormones (insulin)

39. Regulation signaling：
Hormones, Receptors, and
Communication Between Cells

40. • Hormones
– chemical signals that coordinate metabolism
• Hormone Receptors
– Target tissues
– Specific binding
– Types
• Intracellular receptors
• Cell-surface receptors

41. Hormones, Receptors, and
Communication Between Cells
• Intracellular receptors
• lipid soluble hormones
• Steroid hormones, vitamin D, retinoids,
thyroxine
• Bind to intracellular protein receptors
– This binds to regulatory elements by a gene
– Alters the rate of gene transcription
• Induces or represses gene transcription

42. Hormones, Receptors, and Communication Between Cells
Intracellular Receptors

43. Hormones, Receptors, and
Communication Between Cells
• Cell-surface receptors
– Water soluble hormones
• Peptide hormones (insulin), catecholamines,
neurotransmitters
• Three class of cell-surface receptors
– Ligand-Gated Receptors
– Catalytic Receptors
– G Protein-linked Receptors

44. Cell Surface Receptors
Ligand-Gated Receptors
• Binding of a ligand (often a neurotransmitter)
affects flow of ions in/out of cell
• Gamma-amino butyric acid (GABA) binds and
opens chloride channels in the brain
– Valium (anti-anxiety drug) reduces the amount of GABA
required to open the chloride channels

45. Cell-Surface Receptors
Catalytic Receptors
• Binding of hormone activates tyrosine kinase
on receptor which phosphorylates certain
cellular proteins
• Insulin receptor is a catalytic receptor with TYR
Kinase activity

46. Cell-Surface Receptors
G Protein-Linked Receptors
• Binding of hormone
activates an enzyme via a
G-protein communication
link.
• The enzymes produces
intracellular messengers
– Signal transduction
– Second messengers activate
protein kinases

47. Intracellular Messengers:
Signal Transduction Pathways
• Cyclic AMP (cAMP)
• Diacylglycerol (DAG) & Inositol
Triphosphate (IP3)
• Cyclic GMP (cGMP)

48. cAMP is a Second Messenger
• Cyclic AMP is the intracellular agent of
extracellular hormones - thus a ‘second
messenger’
• Hormone binding stimulates a GTP-binding
protein (G protein), releasing
Ga(GTP)
• Binding of Ga(GTP) stimulates adenylyl
cyclase to make cAMP

49. G-Protein-Linked Receptors:
The cAMP Signal Transduction Pathway
• Two types of G-Proteins
• Stimulating G protein (Gs)
– Activate adenylate cyclase
• Inhibitory G proteins (Gi)
– Inhibit adenylate cyclase

50. G Proteins
• G proteins are
trimers
– Three protein units
• Alpha
• Beta
• gamma

51. • Alpha proteins are different in Gs and Gi
– Both have GTPase activity
– Alpha proteins modify adenylate cyclase activity
• AC stimulated by Alpha(s) when activated by a hormone
• AC Inhibited by Alpha(I) when activated by other hormones

52. Family of G Proteins
• Binding of hormones
to receptors causes:
– GTP to displace GDP
– Dissociation of alpha
protein from beta and
gamma subunits
– activation of the alpha
protein
– Inhibition or activation of
adenylate cyclase
– GTPase gradually
degrades GTP and
inactivates the alpha
protein effect (clock)

53. cAMP

54. The cAMP Signal Transduction
Pathway
• cAMP – intracellular messenger
– Elevated cAMP can either activate or inhibit
regulatory enzymes
• cAMP activates glycogen degradation
• cAMP inhibits glycogen synthesis
• [cAMP] affected by rates of synthesis and
degradation
– Synthesis by adenylate cyclase
– Degradation by phosphodiesterase
• Stimulated by insulin
• Inhibited by caffeine

56. What does cAMP do?
Activation of Protein Kinase A by cAMP
• Protein kinase A
– Activates or inhibits several enzymes
– Inactive form: regulatory+catalytic subunits
associated
– Active form: binding of cAMP disassociates
subunits

57. Clinical Case： Cholera
• Severe and rapid diarrheal
disease
– Caused by Vibrio cholerae
– Commonly shock after 4-12 hrs
after first symptoms, death 18 hrs
– several days without
rehydration therapy (subject can
lose up to 20 liters of fluids)
– Source is commonly
contaminated water

58. Cholera
mechanism of action
• V. cholarae produces protein that
attaches to intestinal epithelial cells
– Delivery subunit B (blue) facilitates
entry of subunit A into cell
• Subunit A catalyzes ADP-ribosylation
of the alpha-s subunit of
Gs-protein

59. Clinical Case
• V. cholerae toxin affects alpha-S
subunit
– Inactivates GTPase
– Alpha-S subunit permanently active
• Stimulates adenylate cyclase
– Overproduces cAMP
– stimulates protein kinase
– Phosphorylation of membrane ion
transport proteins – massive losses of Na,
Cl, K, HCO3

60. Hypothetical link to cystic fibrosis
• Cystic fibrosis characterized by
– Salty sweat
– Very thick mucous
• Homozygous genetic defect to chloride transport
to mucous
– Decreased chloride results in less water following due
to osmosis, leading to thicker mucous
• Heterozygous mutation (normal mucous) has
transport protein resistant to effects of cholera
toxin ?

61. Intracellular Messengers:
Signal Transduction Pathways
• Cyclic AMP (cAMP)
• Diacylglycerol (DAG) &
Inositol Triphosphate (IP3)
• Cyclic GMP (cGMP)

62. DAG & IP3
Phosphotidylinositol Signal Transduction Pathway
• Hormone activation of phospholipase C
– Via Gp protein
• Phospholipase C hydrolyzes membrane phospholipids
(phosphotidyl inositol) to produce DAG and IP3
• IP3 stimulates release of Ca from ER
• Protein kinase C activated by DAG and calcium

63. Intracellular Messengers:
Signal Transduction Pathways
• Cyclic AMP (cAMP)
• Diacylglycerol (DAG) & Inositol
Triphosphate (IP3)
• Cyclic GMP (cGMP)

64. cGMP
The cGMP Signal Transduction Pathway
• cGMP effects:
• lowering of blood pressure & decreasing CHD
risk
– Relaxation of cardiac muscle
– Vasodilation of vascular smooth muscle
– Increased excretion of sodium and water by kidney
– Decreased aggregation by platelet cells

65. cGMP
The cGMP Signal Transduction Pathway
• Two forms of guanylate cyclase
• Membrane-bound
• Activated by ANF (atrial natriuretic factor)
– ANF released when BP elevated
• Cytosolic
• Activated by nitric oxide
• NO produced from arginine by NO synthase (activated by
Ca)
– Nitroglycerine slowly produces NO, relaxes cardiac and
vascular smooth muscle, reduces angina
• cGMP activates Protein Kinase G
– Phosphorylates smooth muscle proteins

66. cGMP
The cGMP Signal Transduction Pathway