1. Enzyme Regulation Biochemistry by Reginald Garrett and Charles Grisham
Chapter 15
Enzyme Regulation
Biochemistry by
Reginald Garrett and Charles Grisham

2. Essential Question What are the properties of regulatory enzymes?
How do regulatory enzymes sense the momentary needs of cells?
What molecular mechanisms are used to regulate enzyme activity?

3. Outline of Chapter 15
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?

4. 15.1 – What Factors Influence Enzymatic Activity?
The activity displayed by enzymes is affected by a variety of factors, some of which are essential to the harmony of metabolism
Two of the more obvious ways to regulate the amount of activity are
To increase or decrease the number of enzyme molecule (enzyme level)
To increase or decrease the activity of each enzyme molecule (enzyme activity)

5. A general overview of factors influencing enzyme activity includes the following considerations
Rate depends on substrate availability
Rate slows as product accumulates
Genetic controls (transcription regulation) - induction and repression (enzyme level)
Enzyme activity can be regulated allosterically
Enzyme activity can be regulated through covalent modification
Zymogens, isozymes and modulator proteins may play a role

6. Figure 15.1 Enzyme regulation by reversible covalent modification.

7. Zymogens
Figure 15.2 Proinsulin is an 86-residue precursor to insulin (the sequence shown here is human proinsulin). Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 through 30 (the B chain) remain linked to residues 66 through 87 (the A chain) by a pair of interchain disulfide bridges.

10. Figure 15.4 The cascade of activation steps leading to blood clotting.
Serine protease:
Kallikrein
VIIa
IXa Xa
XIa
XIIa
Thronbin
Figure The cascade of activation steps leading to blood clotting.

11. formation of a blood clot.
Rich in negative charge
formation of a blood clot.

12. Isozymes LDH M is a A4 homotetramer in muscle
LDH H is a B4 homotetramer In heart
Different tissues express different isozyme forms, as appropriate to particular metabolic needs

13. Figure (b) In oxygen-depleted muscle, NAD+ is regenerated in the lactate dehydrogenase reaction.
LDH M works best in pyruvate-to-lactate direction.
LDH H works best in lactate-to-pyruvate direction

14. Modulator proteins are another way that cells mediate metabolic activity
cAMP-dependent protein kinase
Phosphoprotein phosphatase inhibitor-I

15. 15.2 – What Are the General Features of Allosteric Regulation?
Action at "another site"
Allosteric regulation acts to modulate enzymes situated at key steps in metabolic pathways
A  B  C  D  E  F
F, the essential end product, inhibits enzyme 1, the first step in the pathway
This phenomenon is called feedback inhibition or feedback regulation
Enz 1
Enz 2
Enz 3
Enz 4
Enz 5

16. Regulatory enzymes have certain exceptional properties
Their kinetics do not obey the Michaelis-Menten equation
Their v versus [S] plots yield sigmoid- or S-shaped curve
A second-order (or higher) relationship between v and [S]
Substrate binding is cooperative

17. Regulatory enzymes have certain exceptional properties
Their kinetics do not obey the Michaelis-Menten equation
Inhibition of a regulatory enzyme by a feedback inhibitor does not conform to any normal inhibition pattern
Feedback inhibitor (Product) bears the structural similarity to A, may bind to substrate-binding site
If product acts at a site other than the substrate-binding site—called Allosteric inhibition

18. Regulatory enzymes have certain exceptional properties
Their kinetics do not obey the Michaelis-Menten equation
Inhibition of a regulatory enzyme by a feedback inhibitor does not conform to any normal inhibition pattern- Allosteric inhibition
Some effector molecules exert negative effects on enzyme activity, other effectors show stimulatory, or positive, influences on activity
Oligomeric organization
The regulatory effects exerted on the enzyme’s activity are achieved by comformational changes occurring in the protein when effector metabolites bind

19. Symmetry model : Two conformational states
15.3 – Can Allosteric Regulation Be Explained by Conformational Changes in Proteins?
Symmetry model : Two conformational states
Monod, Wyman, Changeux (MWC) Model: allosteric proteins can exist in two states: R (relaxed) and T (taut)
In this model, all the subunits of an oligomer must be in the same state (R or T)
T state predominates in the absence of substrate S
R1  R0  T0  T1
L= T0 / R0
L is equilibrium constant

21. T0  R0 (because R0 becomes R1 , R0 decreases)
Although the relative [R0] concentration is small, S will bind ‘only’ to R0, forming R1
S binds much tighter to R than to T
S-binding drives the conformation transition,
T0  R0 (because R0 becomes R1 , R0 decreases)
Cooperativity is achieved because S binding increases the population of R, which increases the sites available to S
K0.5 (Km)
Ligands such as S are positive homotropic effectors

22. Molecules that influence the binding of something other than themselves are heterotropic effectors
Positive heterotropic effectors or allosteric avtivators (T0  R0)
negative heterotropic effectors or allosteric inhibitors (R0  T0)

23. The notable difference between the MWC and KNF model
The sequential model
Proposed by Koshland, Nemethy, and Filmer (the KNF model) relies on the idea that ligand binding triggers a conformation change in a protein
If the protein is oligomeric, ligand-induced conformation changes in one subunit may lead to conformation changes in adjacent subunits
The notable difference between the MWC and KNF model
In the MWC model, the different conformations have different affinities for the various ligand
the KNF model is based on ligand-induced conformation changes

24. Figure 15.8 The Koshland-Nemethy-Filmer sequential model for allosteric behavior.

25. 15.4 What Kinds of Covalent Modification Regulate the Activity of Enzymes?
Covalent modification 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
Protein kinases and phosphatases work in opposing directions

26. Figure 15.1 Enzyme regulation by reversible covalent modification.

27. Protein kinases phosphorylate Ser, Thr, and Tyr residues in target proteins (Table 15.2)
Phosphorylation introduces a bulky group bearing two negative charges, causing conformational changes that alter the target protein’s function
Protein kinases are classified as Ser/Thr and/or Tyr specific
Kinases are often regulated by intrasteric control

29. Phosphorylation is Not the Only Form of Covalent Modification that Regulates Protein Function
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

32. 15.5 Is the Activity of Some Enzymes Controlled by Both Allosteric Regulation and Covalent Modification?
Glycogen phosphorylase cleaves glucose units from nonreducing ends of glycogen
A phosphorolysis reaction (Fig 15.12)
Regulated both by allosteric control and by covalent modification
Glucose-1-P is converted to glucose-6-P
In muscle, proceeds into glycolysis (chapter 18)
In liver, hydrolysis of glucose-6-P yields glucose

33. Figure 15.12 The glycogen phosphorylase reaction.
Figure The phosphoglucomutase reaction.

34. Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues)
Each subunit contains a pyridoxal phosphate (PLP) cofactor covalently linked (Lys-680)
A tower helix (residues 262 to 278)
An allosteric effector site near the subunit interface
A regulatory phosphorylation site (Ser14)
A glycogen binding site
An active site

35. Glycogen Phosphorylase Activity is Regulated Allosterically
Cooperativity in substrate binding (15.15a)
Inorganic phosphate (Pi) is a positive homotropic effector
ATP and Glucose-6-P are feedback inhibitors, and negative heterotropic effectors (i.e., allosteric inhibitor) (15.15b)
AMP is a positive heterotrophic effector (i.e., allosteric activator) (15.15c)
ATP and AMP bind to the same site (reciprocal regulation in the cellular concentration of ATP and AMP

36. Figure 15.14 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.

37. MWC model The active form of the enzyme is designated the R state
The inactive form of the enzyme is denoted as 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 change occurs at the subunit interface between the T (Asp283) and R state (Arg569) –Pi
This conformational change at the subunit interface is linked to a structural change at the active site that affects catalysis (fig 15.17)

38. Figure  The mechanism of covalent modification and allosteric regulation of glycogen phosphorylase. The T states are blue and the R states blue-green.

39. 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 (Figure 15.16)
Phosphorylation of Ser14 causes a dramatic conformation change in phosphorylase (Figure 15.17)

40. Figure 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. This phosphorylation is mediated by an enzyme cascade
Cyclic AMP (cAMP) 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

42. Figure The hormone-activated enzymatic cascade that leads to activation of glycogen phosphorylase.

43. Figure  The adenylyl cyclase reaction yields 3',5' -cyclic AMP and pyrophosphate. The reaction is driven forward by subsequent hydrolysis of pyrophosphate by the enzyme inorganic pyrophosphatase.

44. 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.

45. A classic example of allostery
Hemoglobin
A classic example of allostery
Hemoglobin (Hb) and myoglobin (Mb) are oxygen transport and storage proteins
Compare the oxygen binding curves for hemoglobin and myoglobin
Myoglobin is monomeric; hemoglobin is tetrameric
Mb: 153 residues
Hb: two as of 141 residues, 2 bs of 146 (a2b2)

47. Hemoglobin Function Hb must bind oxygen in lungs and release it in capillaries
Adjacent subunits' affinity for oxygen increases
This is called positive cooperativity

48. Figure 15.21 O2-binding curves for hemoglobin and myoglobin.  

49. Competition between oxygen and H+
The Bohr Effect
Competition between oxygen and H+
Discovered by Christian Bohr
Binding of protons diminishes oxygen binding
Binding of oxygen diminishes proton binding
Important physiological significance

50. Figure  The oxygen saturation curves for myoglobin and for hemoglobin at five different pH values: 7.6, 7.4, 7.2, 7.0, and 6.8.

51. Carbon dioxide diminishes oxygen binding
Bohr Effect II
Carbon dioxide diminishes oxygen binding
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

52. The ionic binding of BPG to the two β-subunits of Hb
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.

53. Fetal Hb differs from adult Hb – with γ-chains in place of β-chains – and thus a α2γ2 structure
Fetal Hemoglobin Has a Higher Affinity for O2 Because it has a Lower Affinity for BPG