1. Chapter 15 Enzyme Regulation

2. Outline What factors influence enzymatic activity ?
What are the general features of allosteric regulation ?
Can allosteric regulation be explained by conformational changes in proteins ?
What kinds of covalent modification regulate the activity of enzymes ?
Is the activity of some enzymes controlled by both allosteric regulation and covalent modification ?
Special focus: is there an example in nature that exemplifies the relationship between quaternary structure and the emergence of allosteric properties ? (hemoglobin and myoglobin – paradigms of protein structure and function).

3. 15.1 – What Factors Influence Enzymatic Activity?
The availability of substrates and cofactors usually determines how fast the reaction goes. As product accumulates, the apparent rate of the enzymatic reaction will decrease due to lack of substrate.
Enzyme activity can be regulated through covalent modification.
Enzyme activity can be regulated allosterically.
Zymogens, isozymes, and modulator proteins may play a role.
Genetic regulation of enzyme synthesis and decay determines the amount of enzyme present at any moment.

4. Levels of Enzyme Regulation
1. Enzyme level: The enzyme may be activated or inhibited by either noncovalent or covalent interactions. This is the most rapid control system.
2. Hormonal level: A hormone is secreted and carries a message to the cell and in turn an enzyme is activated or inhibited. The speed of this control system is intermediate.
3. Gene level: A message is sent to the nucleus either express or repress a gene. This determines the amount of enzyme produced and is the slowest control measure.

5. 15.1 – What Factors Influence Enzymatic Activity?
Figure Enzyme regulation by reversible covalent modification. Depending on the enzyme, phosphorylation may activate or inactivate its catalytic function.

6. 15.1 – What Factors Influence Enzymatic Activity?
Zymogens or proenzymes are inactive precursors of enzymes.
Typically, proteolytic cleavage produces the active enzyme.
Figure 15.2
Proinsulin is an 86-residue precursor to insulin

7. Figure 15.3 The proteolytic activation of chymotrypsinogen

8. Proteolytic Enzymes of the Digestive Tract

9. The Cascade of Activation Steps Leading to Blood Clotting
Figure 15.4
The intrinsic and extrinsic pathways converge at factor X, and the final common pathway involves the activation of thrombin and its conversion of fibrinogen into fibrin, which aggregates into ordered filamentous arrays that become crosslinked to form the clot.

10. Isozymes Are Enzymes With Slightly Different Subunits
Figure The isozymes of lactate dehydrogenase (LDH).

11. 15.2 – What Are the General Features of Allosteric Regulation?
Action at "another site"
Allosteric enzymes have quaternary structure and regulation is at a site other than the active site.
Enzymes situated at key steps in metabolic pathways are modulated by allosteric effectors.
The effectors are usually produced elsewhere in the pathway (heterotropic). S or P = homotropic.
Effectors may be feed-forward activators or feedback inhibitors.
Kinetics are sigmoid ("S-shaped"), see MM plot.

12. 15.2 – What Are the General Features of Allosteric Regulation?
Effects
A positive effector activates the enzyme (an activator).
A negative effector inhibits the enzyme (an inhibitor).
Positive cooperativity increases substrate binding in an adjacent subunit.
Negative cooperativity decreases substrate binding in an adjacent subunit.

13. 15.2 – What Are the General Features of Allosteric Regulation?
Figure Sigmoid v versus [S] plot.
The dotted line represents the hyperbolic plot characteristic of normal Michaelis-Menten kinetics.

14. 15.3 Allosteric Regulation and Conformational Changes in Subunits.
Monod, Wyman, Changeux (MWC) Model: allosteric proteins can exist in two states: R (relaxed) and T (taut or tight).
In this two-state model, all the subunits of an oligomer must be in the same state (they all change together) and is therefore termed the concerted model.
T state predominates in the absence of substrate S.
S binds much tighter to R than to T.

15. The Concerted Model for Allosteric Regulation (MWC)
Figure Allosteric effects:
A and I binding to R and T, respectively.

16. The Concerted Model for Allosteric Regulation
Figure 15.7
Allosteric effects: A and I binding to R and T, respectively.

17. The Concerted Model for Allosteric Regulation
Figure Allosteric effects:
A and I binding to R and T, respectively.

18. More about the MWC model
Positive cooperativity is achieved because S binding increases the population of R, which increases the sites available to S.
In the MWC ligands such as S are positive homotropic effectors.
Molecules that influence the binding of something other than themselves are heterotropic effectors.
The MWC model does not explain negative cooperativity.

19. The Sequential Model for Allosteric Regulation (KNF)
An alternative model – proposed by Koshland, Nemethy, and Filmer (the KNF model) relies on the idea that ligand binding triggers a conformation change in a protein.
In this one-state model, ligand-induced conformation changes in one subunit may lead to conformation changes in adjacent subunits.
The KNF model explains how ligand-induced conformation changes could cause subunits to adopt conformations with little affinity for the ligand – i.e., negative cooperativity.
The KNF model is termed the sequential model.

20. The Sequential Model for Allosteric Regulation
Figure 15.8
The Koshland-Nemethy-Filmer sequential model for allosteric behavior. (a) S binding can, by induced fit, cause a conformation change in the subunit to which it binds. (b) If subunit interactions are tightly coupled, binding of S to one subunit may cause the other subunit to assume a conformation having a greater or lesser affinity for S. That is, the ligand-induced conformational change in one subunit can affect the adjoining subunit.

21. The Sequential Model for Allosteric Regulation
Figure 15.8
The Koshland-Nemethy-Filmer model.
Theoretical curves for the binding of a ligand to a protein having four identical subunits, each with one binding site for the ligand.

22. Allosteric Models

23. 15.4 Covalent Modification Regulate the Activity of Enzymes
Enzyme activity can be regulated through reversible phosphorylation.
This is the most prominent form of covalent modification in cellular regulation.
Phosphorylation is accomplished by protein kinases.
Each protein kinase targets specific proteins for phosphorylation.
Phosphoprotein phosphatases catalyze the reverse reaction – removing phosphoryl groups from proteins.
Kinases and phosphatases themselves are targets of regulation.

24. 15.4 Covalent Modification Regulate the Activity of Enzymes
Enzymes that catalyze phosphate transfer
Kinase:
An enzyme that transfers phosphate to ADP or AMP or from ATP.
Phosphatase or phosphoprotein phosphatase:
An enzyme that hydrolyzes a phosphate off of a substrate.
Phosphorylase:
An enzyme that adds a phosphate to substrate but does not use ATP to do so.

25. 15.4 Covalent Modification Regulate the Activity of Enzymes
Protein kinases phosphorylate Ser, Thr, and Tyr residues in target proteins.
Kinases typically recognize specific amino acid sequences in their targets.
In spite of this specificity, all kinases share a common catalytic mechanism based on a conserved core kinase domain of about 260 residues, Fig
Kinases are often regulated by intrasteric control, in which a regulatory subunit (or domain) has a pseudosubstrate sequence that mimics the target sequence, minus the Ser or Thr that is needed. Such is the case in the R2C2 tetramer of PKA.

26. 15.4 Covalent Modification Regulate the Activity of Enzymes

27. 15.4 Covalent Modification Regulate the Activity of Enzymes
Figure 15.9
Protein kinase A is shown complexed with a pseudosubstrate peptide (orange).
This complex also includes ATP (red) and two Mn2+ ions (yellow) bound at the active site.

28. Cyclic AMP-dependent protein kinase
Figure cyclic AMP-dependent protein kinase, also known as protein kinase A (PKA), is a 150- to 170-kD R2C2 tetramer in mammalian cells.
The two R (regulatory) subunits bind cAMP; cAMP binding releases the R subunits from the two C (catalytic) subunits. C subunits are enzymatically active as monomers.

29. Other Covalent Modification of Protein
Several hundred different chemical modifications of proteins have been discovered.
Only a few of these are used to achieve metabolic regulation through reversible conversion of an enzyme between active and inactive forms.
A few are summarized in Table 15.3.
Three of the modifications in Table 15.3 require nucleoside triphosphates (ATP, UTP) that are related to cellular energy status.

30. Other Covalent Modification of Protein

31. 15.5 Enzymes Controlled by Both Allosteric and Covalent Regulation?
Glycogen phosphorylase (GP) is an example of the many enzymes that are regulated both by allosteric controls and by covalent modification.
GP uses Pi to attack glucose at an α(1-4) linkage on the nonreducing ends of glycogen.
This converts glycogen into readily usable fuel in the form of glucose-1-phosphate.
This is a phosphorolysis reaction.
Muscle GP is a dimer of identical subunits, each with PLP covalently linked.
There is an allosteric effector site at the subunit interface.

32. Glycogen Phosphorylase
Figure The glycogen phosphorylase reaction converts
glycogen into readily usable fuel in the form of glucose-1-P.
The enzyme cannot cleave an α(1-6) branchpoint.

33. Phosphoglucomutase
Figure The phosphoglucomutase reaction converts
glucose-1-P into the glycolytic substrate, glucose-6-P.

34. The structure of glycogen phosphorylase
Glycogen phosphorylase is a dimer of identical subunits each having 842 residues.
Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface.
A regulatory phosphorylation site is located at Ser14 on each subunit.
A glycogen-binding site exerts regulatory control.
Each subunit contributes a “tower helix” (residues 262 to 278) to the subunit-subunit interface.
In the dimer, the tower helices extend from their respective subunits and pack against each other.

35. The structure of glycogen phosphorylase
Figure 15.13
The structure of glycogen phosphorylase.
One monomer of the homodimer.

36. Glycogen Phosphorylase Activity is Regulated Allosterically
Muscle glycogen phosphorylase shows cooperativity in substrate binding.
ATP and glucose-6-P are allosteric inhibitors of glycogen phosphorylase.
AMP is an allosteric activator of glycogen phosphorylase.
When ATP and glucose-6-P are abundant, glycogen breakdown is inhibited.
When cellular energy reserves are low (i.e., high [AMP] and low [ATP] and [G-6-P]) glycogen catabolism is stimulated.

37. Glycogen Phosphorylase Activity is Regulated Allosterically
Figure v versus S curves for glycogen phosphorylase.
The response to the concentration of the substrate phosphate (Pi).
ATP is a feedback inhibitor.
AMP is a positive effector. It binds at the same site as ATP.

38. Glycogen phosphorylase conforms to the MWC model
The active form of the enzyme is designated the R state.
The inactive form of the enzyme is denoted the T state.
AMP promotes the conversion to the active state.
ATP, glucose-6-P, and caffeine favor conversion to the inactive T state.
A significant conformation change occurs at the subunit interface between the T and R state.
This conformational change at the interface is linked to a structural change at the active site that affects catalysis.

39. Glycogen Phosphorylase is Controlled Both Allosterically and Covalently
Figure 15.15
The mechanism of covalent modification and allosteric regulation of glycogen phosphorylase.
Favored
Favored

40. A Conformation Change Regulates Activity of Glycogen Phosphorylase
Figure 15.16
The major conformational change that occurs in the N-terminal residues upon phosphorylation of Ser14. Ser14 is shown in red.
N-terminal conformation of phosphorylated enzyme (phosphorylase a): yellow.
N-terminal conformation of unphosphorylated enzyme (phosphorylase b): cyan.

41. Regulation of GP by Covalent Modification
In 1956, Edwin Krebs and Edmond Fischer showed that a ‘converting enzyme’ could convert phosphorylase b to phosphorylase a.
Three years later, Krebs and Fischer show that this conversion involves covalent phosphorylation.
The enzyme is phosphorylase kinase and this phosphorylation is mediated by an enzyme cascade (Figure 15.17).

42. Glycogen phosphoryase is activated by a cascade of reactions
Figure The hormone-activated enzymatic cascade that leads to activation of glycogen phosphorylase.

43. The Adenylyl Cyclase Reaction
Figure The adenylyl cyclase reaction. The reaction is driven forward by subsequent hydrolysis of pyrophosphate by the enzyme inorganic pyrophosphatase.

44. The Adenylyl Cyclase Reaction
(Shown here are the products of the reaction. ATP, the reactant, is shown on the previous slide.)
Figure The adenylyl cyclase reaction. The reaction is driven forward by subsequent hydrolysis of pyrophosphate by the enzyme inorganic pyrophosphatase.

45. 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 G(GTP).
Binding of G(GTP) stimulates adenylyl cyclase to make cAMP.

46. cAMP is a Second Messenger
Figure Hormone binding to its receptor leads via G- protein activation to cAMP synthesis. Adenylyl cyclase and the hormone receptor are integral membrane proteins; Gα and Gβγ are membrane-anchored proteins.

47. Modes of regulation Zymogens or proenzymes. Enzyme cascades. Isozymes.
Allosterism
Covalent modification.
Second messengers.
Combined effects

48. A classic example of allostery
Hemoglobin
A classic example of allostery
Hemoglobin is an oxygen transport protein and myoglobin is an O2 storage protein.
Look at the oxygen binding curves for hemoglobin and myoglobin.
Myoglobin is monomeric; hemoglobin is tetrameric, α2β2 .
Mb: 153 aa, 17,200 MW.
Hb: two α chains of 141 residues, 2 β chains of 146 residues.

49. Figure 15.20 O2-binding curves for hemoglobin and myoglobin
Myoglobin P50 = 2.8 torr and Hemoglobin P50 = 26 torr.

50. The structure of myoglobin is similar to that of the Hb monomer
Figure 15.21
The myoglobin and hemoglobin structures.
Each is a protein with a heme (aromatic).
Myoglobin is monomeric.
Hemoglobin is tetrameric.

51. Mb and Hb use porphryins to bind Fe2+
Figure 15.22
Heme is formed when protoporphyrin IX binds Fe2+

52. Fe2+ is coordinated by His F8
Iron interacts with six ligands in Hb and Mb.
Four of these are the N atoms of the porphyrin.
A fifth ligand is donated by the imidazole side chain of amino acid residue His F8.
(This residue is on the sixth or “F” helix, and it is the 8th residue in the helix, thus the name.).
When Mb or Hb bind oxygen, the O2 molecule adds to the heme iron as the sixth ligand.
The O2 molecule is tilted relative to a perpendicular to the heme plane.

53. Fe2+ is coordinated by His F8
Figure 15.23
The six liganding positions of an iron atom in Hb and Mb.

54. Myoglobin Structure Mb is a monomeric heme protein.
Mb polypeptide "cradles" the heme group.
Fe in Mb is Fe2+ - ferrous iron - the form that binds oxygen.
Oxidation of Fe yields 3+ charge - ferric iron.
Mb with Fe3+ is called metmyoglobin and does not bind oxygen.

55. O2 Binding Alters Mb Conformation
In deoxymyoglobin, the ferrous ion actually lies nm above the plane of the heme.
When oxygen binds to Fe in heme of Mb, the heme Fe is drawn toward the plane of the porphyrin ring.
With oxygen bound, the Fe2+ atom is only nM above the plane.
For Mb, this small change has little consequence.
But a similar change in Hb initiates a series of conformational changes that are transmitted to adjacent subunits.

56. Hb Has an α2β2 Tetrameric Structure
Figure 15.24
An αβ dimer of Hb, with packing contacts indicated in blue.
The sliding contacts made with the other dimer are shown in yellow. The changes in these sliding contacts are shown in Figure

57. Cooperative Binding of Oxygen Influences Hemoglobin Function
Mb, an oxygen storage protein, has a greater affinity for oxygen at all oxygen pressures.
Hb is different – it must bind oxygen in lungs and release it in capillaries.
Hb becomes saturated with O2 in the lungs, where the partial pressure of O2 is about 100 torr.
In capillaries, pO2 is about 30 torr, and oxygen is released from Hb.
The binding of O2 to Hb is cooperative – binding of oxygen to the first subunit makes binding to the other subunits more favorable.

58. O2 binding curves of Mb and Hb
The oxygen binding curve of Mb resembles an enzyme:substrate saturation curve.

59. An Alternative O2 Binding Curve for Hb
Oxygen saturation curve for Hb in the form of Y versus pO2 assuming n = 4 and P50 = 26 torr.
Y is the fractional saturation of Hb.

60. An Alternative O2 Binding Curve for Hb
A comparison of the experimentally observed O2 curve for Hb yielding a value for n of 2.8,
the hypothetical curve if n = 4,
and the curve if n = 1 (non-interacting O2-binding sites).

61. The Conformation Change
The secret of Mb and Hb
Oxygen binding changes the Mb conformation.
Without oxygen bound, Fe2+ is out of heme plane.
Oxygen binding pulls the Fe2+ into the heme plane.
Fe2+ pulls its His F8 ligand along with it.
The F helix moves when oxygen binds.
Total movement of Fe2+ is nm – i.e., 0.29 Å.
This change means little to Mb, but lots to Hb!

62. Oxygen Binding by Hb Induces a Quaternary Structure Change
When deoxy-Hb crystals are exposed to oxygen, they shatter. This is evidence of a large-scale structural change.
One alpha-beta pair moves relative to the other by 15 degrees upon oxygen binding.
This massive change is induced by movement of Fe by nm when oxygen binds.

63. Oxygen binding to Hb results in a 15° rotation of one αβ pair relative to the other
Figure Subunit motion in hemoglobin when the molecule goes from the (a) deoxy form to the (b) oxy form.

64. Fe2+ Movement by Less Than 0
Fe2+ Movement by Less Than 0.04 nm Induces the Conformation Change in Hb
In deoxy-Hb, the iron atom lies out of the heme plane by about 0.06 nm.
Upon O2 binding, the Fe2+ atom moves about nm closer to the plane of the heme.
As if the O2 is drawing the heme iron into the plane.
This may seem like a trivial change, but its biological consequences are far-reaching.
As Fe2+ moves, it drags His F8 and the F helix with it.
This change is transmitted to the subunit interfaces, where conformation changes lead to the rupture of salt bridges.

65. Fe2+ Movement by Less Than 0
Fe2+ Movement by Less Than 0.04 nm Induces the Conformation Change in Hb
Figure 15.26
Changes in the position of the heme iron atom upon oxygenation lead to conformational changes in the hemoglobin molecule.

66. Salt bridges that stabilize deoxy-Hb are broken in oxy-Hb
Figure 15.27
Salt bridges between different subunits in human deoxy-Hb. These noncovalent, electrostatic interactions are disrupted upon oxygenation.
The salt bridges and H-bonds involving interactions between N-terminal and C-terminal residues in the α-chains.
The salt bridges and H bonds involving C-terminal residues of β-chains

67. The Physiological Significance of the Hb:O2 Interaction
Hb must be able to bind oxygen in the lungs.
Hb must be able to release oxygen in capillaries.
If Hb behaved like Mb, very little oxygen would be released in capillaries - see Figure 15.20!
The sigmoid, cooperative oxygen binding curve of Hb makes its physiological actions possible!
Hb exhibits properties of both the MWC and KNF models.
In the T state only the α subunits can bond O2 and in the R state all subunits can bind O2.

68. H+ Promotes Dissociation of Oxygen from Hemoglobin
Binding of O2 to Hb is affected by several agents, including H+, CO2, 2,3-bisphosphoglycerate and chloride ions.
The effect of H+ is particularly important. This is the Bohr effect.
Deoxy-Hb has a higher affinity for H+ than oxy-Hb.
Thus, as pH decreases, dissociation of O2 from hemoglobin is enhanced.
Ignoring the stoichiometry of O2 and H+, we can write:

69. H+ Promotes Dissociation of Oxygen from Hemoglobin
Figure 15.28
The oxygen saturation curves for myoglobin and for hemoglobin at five different pH values: 7.6, 7.4,7.2, 7.0, 6.8.

70. The Antagonism of O2 Binding by H+ is Termed the Bohr Effect
The effect of H+ on O2 binding was discovered by Christian Bohr (the father of Neils Bohr, the atomic physicist).
Binding of protons diminishes oxygen binding.
Binding of oxygen diminishes proton binding.
Important physiological significance.
HHbO2 <==> HbO2 + H+ pKa = 6.6
HHb <==> Hb + H+ pKa = 8.2

71. Figure 15.20 O2-binding curves for hemoglobin and myoglobin
Myoglobin P50 = 2.8 torr and Hemoglobin P50 = 26 torr.

72. CO2 Also Promotes the Dissociation of O2 from Hemoglobin
Carbon dioxide diminishes oxygen binding
1. Hydration of CO2 in tissues and extremities leads to proton production:
These protons are taken up by Hb as oxygen dissociates.
The reverse occurs in the lungs.
2. CO2 also binds covalently to the N-terminus. And fovors the T state.

73. Summary of the Physiological Effects of H+ and CO2 on O2 Binding by Hemoglobin
At the tissue-capillary interface, CO2 hydration and glycolysis produce extra H+, promoting additional dissociation of O2 where it is needed most.
At the lung-artery interface, bicarbonate dehydration (required for CO2 exhalation) consumes extra H+, promoting O2 binding.

74. 2,3-Bisphosphoglycerate
An Allosteric Effector of Hemoglobin
In the absence of 2,3-BPG, oxygen binding to Hb follows a rectangular hyperbola!
The sigmoid binding curve is only observed in the presence of 2,3-BPG.
Since 2,3-BPG binds at a site distant from the Fe where oxygen binds, it is called an allosteric effector.

75. BPG Binding to Hb Has Important Physiological Significance
Figure The structure, in ionic form of BPG or 2,3-bisphosphoglycerate, an important allosteric effector of Hb.

76. BPG Binding to Hb Has Important Physiological Significance
The "inside" story......
Where does 2,3-BPG bind ?
"Inside“.
in the central cavity of the tetramer.
What is special about 2,3-BPG ?
Negative charges interact with 2 Lys, 4 His, 2 N-termini.
Fetal Hb - lower affinity for 2,3-BPG, higher affinity for oxygen, so it can get oxygen from mother.

77. BPG Binding to Hb Has Important Physiological Significance
Figure 15.31
The ionic binding of BPG to the two β-subunits of Hb. BPG lies at the center of the cavity between the two β-subunits.
The resulting conformational change precludes O2 binding.

78. CO2 Also Promotes the Dissociation of O2 from Hemoglobin
Figure 15.29
Oxygen binding curves of blood and of hemoglobin in the absence and presence of CO2 and BPG.

79. Fetal Hemoglobin Has a Higher Affinity for O2 Because it has a Lower Affinity for BPG
The fetus depends on its mother for O2, but its circulatory system is entirely independent.
Gas exchange takes place across the placenta.
Fetal Hb differs from adult Hb – with γ-chains in place of β-chains – and thus a α2γ2 structure.
As a result, fetal Hb has a higher affinity for O2.
Why does fetal Hb bind O2 more tightly ?
Fetal γ-chains have Ser instead of His at position 143 and thus lack two of the positive charges in the BPG binding cavity
BPG binds less tightly and Hb F thus looks more like Mb in its O2 binding behavior.

80. Fetal Hemoglobin Has a Higher Affinity for O2 Because it has a Lower Affinity for BPG
Figure Comparison of the oxygen saturation curves of Hb A and Hb F under similar conditions of pH and [BPG].

81. Sickle-Cell Anemia is a Molecular Disease
Sickle-cell anemia patients have abnormally-shaped red blood cells.
The erythrocytes are crescent-shaped instead of disc-shaped.
The sickle cells pass less freely through the capillaries, impairing circulation and causing tissue damage.
A single amino acid substitution in the β-chains of Hb causes sickle-cell anemia.
Glu at position 6 of the β-chains is replaced by Val.
As a result, Hb S molecules aggregate into long, chainlike polymeric structures.

82. Hemoglobin and Nitric Oxide
Nitric oxide (NO·) is a simple gaseous molecule that acts as a neurotransmitter and as a second messenger in signal transduction (see Chapter 32).
NO· is a high-affinity ligand for Hb, binding to the heme iron 10,000 times more tightly than O2.
So why is NO· not bound instantaneously to Hb, preventing its physiological effects ?
NO· reacts with the –SH of Cys93β, forming an S-nitroso derivative:

83. Hemoglobin and Nitric Oxide
The S-nitroso group is in equilibrium with other S-nitroso compounds formed by reaction of nitric oxide with small-molecule thiols such as free Cys or glutathione:
These small-molecule thiols transfer NO· from erythrocytes to endothelial receptors, where it exerts its physiological effects.

84. End Chapter 15 Enzyme Regulation