1. Piamsook Pongsawasdi Oct 2017
Metabolic regulation
Advanced Biochemistry
Piamsook Pongsawasdi
Oct 2017

2. OVERVIEW OF METABOLISM REGULATION OF METABOLIC PATHWAYS
PART I
OVERVIEW OF METABOLISM
REGULATION OF METABOLIC PATHWAYS

3. Metabolism
All of the chemical reactions in a cell
- catabolism
- anabolism
sum characteristics of a cell in an environment
Intricately regulated at enzyme and gene levels which govern life and cellular adaptation to environment.

4. Transformation of nutrients Energy transformations
Metabolism involves:
Transformation of nutrients
Energy transformations
Synthesis and degradation processes
Excretion of waste products
Glycolysis and TCA cycle are central core of metabolism
All reactions are enzyme catalyzed.

5. Catabolism
Breakdown of complex molecules into simpler molecules
building blocks
intermediates
1. Release energy
2. Oxidative process
NAD+ and FAD collects electron and converts to NADH and FADH2
3. Convert to few end products

6. Anabolism
Build complex molecules from simpler molecules
e.g. Amino acids proteins
1. Consume energy
2. Reductive process (provides electron by NADPH)
3. Diverge to many end products

7. Amphibolic pathway
• Intermediary pathway that serves both in catabolism and anabolism
Anaplerotic reactions • Reactions that replenish depleted interme-diates, e.g. Pyr Carboxylase replenishes OAA. Replenished reaction can be by breakdown of amino acids (transamination/deamination) or fatty acids.

8. Amphibolic pathway of TCA cycle

9. Summary of metabolism
Metabolism is composed of interconnecting reactions and pathways
Thermodynamics determine the direction of the pathways
ATP is the universal currency
Oxidation of carbon fuel is major source of cellular energy
Metabolic pathways are tightly regulated

10. All pathways intertwined in a multidimensional network of reactions
Pathways compose of sequential reactions: Linear, Branch, or Cyclic

11. Summary of metabolism (continued)
Metabolic pathways are irreversible at some steps
(to be spontaneous - overall ΔG of the pathway is negative)
Every pathway has a committed step
(usually the first irreversible step unique to the pathway)
Synthetic and degradative pathways differ, they are distinct by compartmentation or bypass reaction

12. Synthetic and degradative pathways don’t happen at the same time
They can share some common steps but they are never simply the reverse of one another 
provides separate control of the processes (at irreversible step)
Synthetic pathways always use more ATP than a degradative pathway will produce.
If both synthetic and degradative pathways occurred at the same time, “wasteful” hydrolysis of ATP would result.
This is termed a “futile cycle.”

13. Irreversible pathway provides separate control
x y C D Ea Em Eb En Ec Ed

14. The Nature and Significance of Metabolic Regulation
conservation of energy and materials
maintenance of metabolic balance despite changes in environment
homeostasis (regulation of steady state e.g. [blood glucose] is held nearly constant at 5 mM) Failure to maintain diseases.
primarily concern with the control of metabolic flux through a pathway to maintain steady state (cells and organisms exist in a dynamic steady state, that is [s] remains constant though the rate of metabolic flow (v) may be high and variable
A S P v1 v2

15. Metabolic flux
The rate of material processing through a metabolic pathway
Flux = flow of metabolites through a pathway
- quantitatively predict system behaviour as a response to any external signal(s)

16. Metabolism is driven by enzymes Enzyme reaction
E + S ES E + P
Major factors affecting product formation are:
1. Substrate concentration
2. Enzyme concentration
3. Enzyme activity

17. Enzyme regulation is a key factor in metabolic regulation
Level of enzyme regulation
• Enzyme concentration
regulated by alteration of
- transcription
- translation
- degradation
• Enzyme activity
- post-translation

18. Enzyme Activity is affected by many factors
Step 1-5 change in [E]
Step 6-10 change in activity

19. Protein Turnover

20. Compartmentation : Segregation of substrate and enzyme
Specific transporter facilitates substrate accessibility

21. All enzymes are sensitive to [S]

22. Summary of steps of regulation

23. Two Types of Enzyme-catalyzed reactions in cells
E + S ES E + P
Cellular reactions can be divided into
a. Equilibrium or near equilibrium reaction(step2,3)
b. Non-equilibrium reaction (step 1)
Enzymes that catalyze reaction which is far from equilibrium are more important for regulation than those that catalyze near equilibrium reactions.

24. Equilibrium or near-equilibrium reaction
- enzyme is in sufficient quantity that it converts substrate into product very fast
- flux through this kind of reactions is regulated by the availability of the substrate and the removal of the product, not by regulating the enzyme activity E1
A + B C
enzyme activity relative to Km
- easily switch direction depending on the concentration of substrate and product (law of mass action)

25. Effect of [substrate]
• rate increases as [substrate] increases
• no further increase occurs after all enzyme molecules are saturated with substrate

26. Km values for enzymes are typically just above the concentrations of substrates in cells (pink area) so that enzyme velocities are relatively sensitive to physiological changes in substrates and will likely adjust in velocity to match changes in supply of substrate.

27. = -RT lnK′eq

28. To identify equilibrium or non-equilibrium reactions
Mass action ratio Q = [C][D] / [A][B]
When Q and K′eq are within 1 to 2 orders of magnitude of each other, the reaction is near equilibrium
e. g. 6 of the 10 steps in the glycolytic pathway (Table 15-3)

30. Non-equilibrium reaction
E E E E E5
A B C D E F
- regulated by allosteric enzyme, (E3) have insufficient activity (acts as a dam)
- reactants accumulate due to the upstream reaction (of the non-equilibrium reaction) and downstream reaction drained the products
- regulation of this enzyme (E3) activity determine the overall flux of the pathway
- irreversible reaction, important in metabolic control

31. Regulatory Enzymes (allosteric enzyme)
catalyze the rate-limiting steps in pathways (tend to catalyze very exergonic reactions that are essentially irreversible)
often located at early step of the pathway, or at branch points where the substrate could enter multiple pathways
Work at steady state condition,S-curve kinetics

32. Equilibrium versus Steady State
no net flux in any direction
A B
Steady state
constant flux in one direction
A B C

33. Adenine nucleotides play special roles in metabolic regulation
Maintaining constant [ATP] is so important for cells
[ATP] in a typical cell of animal tissues is  5 mM, and many ATP-using enzymes have Km  mM
If ATP drops, these E are not saturated with ATP→ reaction rates ↓, cells cannot survive this kinetic effect on so many reactions

34. Thermodynamic effect of lowered [ATP]
To get cellular work, ATP is converted to ADP or AMP, the ATP/ADP or ATP/AMP has important effect on all reactions that use these cofactors
[AMP] is a more sensitive indicator of an energetic state of cells than [ATP]. In the cell, [ATP] (5mM) is far higher than [AMP] (0.1 mM). When ATP is used (e.g. muscle contraction), AMP is produced by ATP→ADP, then 2ADP AMP + ATP
If [ATP] ↓ 10%, increase in [AMP] is greater than that of ADP
Adenylate kinase

35. Effect of enzyme on metabolic flux can be determined
HK,PFK,PI are added to rat liver homogenate, then measure F1,6BP formed from glucose
HK and PFK affect flux
Effect of HK is more
Regulatory vs Control
Mechanism
HK control glycolytic flux
PFK maintain metabolite homeostasis

36. Another example of Regulatory vs Control Mechanism: Flux towards glycogen synthesis from glucose
Capillary
In muscle, insulin acts to
1. ↑ glucose transport in
2. Induce HK synthesis
3. Activate glycogen synthase by phosphory-lation (frame 115)
Step 1&2- ↑ glucose flux
→Metabolic Control
Step 3-maintain [metabo-lite],→MB Regulation

37. How to regulate enzyme activity?(short-term control)
The key to regulate enzyme activity is conformational change.
Changes in structure can have profound changes on the function or activity of a protein molecule
Conformational changes can occur in response to interactions with other molecules, such as
Substrate, substrate analogs
Allosteric modulators
Covalently added groups (triggers by hormone, signal transducer)
Regulatory subunits or accessory proteins
Proteolytic enzymes

38. Allosteric Regulation : a mean to regulate enzyme activity

39. Conformation change due to allosteric control

40. Allosteric regulation
Enable enzyme to rapidly adjust to signals
Velocity of the enzyme (v) is
- rather insensitive to change of substrate concentration.
- sensitive to change of amount of effector/ modulator (inhibitor or activator)
The v vs. [S] plot is sigmoidal : a consequence of co-operative system.
Cooperativity could either be + or –
Allosteric enzyme is usually multimeric and has two or more conformations (active vs inactive)
Catalyzes reaction at committed step or first step of a branch pathway

41. Feedback inhibition
Feedforward activation

42. Feedback inhibition (End product inhibition)
inhibition of one or more critical enzymes in a pathway regulates entire pathway
each end product
regulates its
own branch
of the pathway
each end product
regulates the
initial pacemaker
enzyme*
*catalyzes the slowest or rate-limiting reaction in the pathway

43. Example of feedback inhibition
Specific, in that
Isoleucine inhibits only E1 – not any of the other enzymes (E2 – E5)
Only isoleucine inhibits E1

44. Feed forward activation

45. Kinetics of cooperativity of hemoglobin

46. Kinetics of allosteric enzyme
A sigmoid rather than hyperbolic curve for a plot of vo vs. [S] suggests allosteric, or cooperative interactions among subunits
Modulating activators and/or inhibitors can cause major shifts in these curves
Cooperativity can be :
Homotropic : when S is also A (e.g. O2 for Hb case)
Heterotropic : when M is not S, M can be A (positive cooperativity) or I (negative cooperativity)

47. Kinetics of allosteric control
(R-state)
(R state)
(T-state)
(T-state)

48. Example of kinetics of allosteric enzyme : Glucokinase

49. Responses of allosteric enzymes to positive (+) and negative (-) modulators
K0.5 changes, no change in Vmax
Vmax changes, K0.5 is nearly constant, rare case

50. Conformation of allosteric enzyme
Enzyme exists in 2 conformation states:
a. R-state or relaxed state
(active conformation)
b. T-state or tense state
(inactive conformation)

51. Two models of cooperativity
concerted model,
all subunits are either in the R or the T state, and S binds only to the R state.
(b) sequential model, binding of S to a subunit converts only that subunit to the R conformation. Neighboring subunits might remain in the T state or might assume conformations between T and R.

52. Mechanism and Example of Allosteric Effect
Kinetics
Cooperation
Models
Allosteric site
R = Relax
(active)
[S] vo
Homotropic
(+)
Concerted
(+)
Allosteric site
[S] vo
(+) A
Heterotropic
(+)
Sequential
(+) X
T = Tense
(inactive)
[S] vo I
Heterotropic
(-)
Concerted
(-)
(-) X X
Juang RH (2004) BCbasics

53. Example of allosteric enzyme : Aspartate transcarbamoylase (ATCase)
Regulates first step of pyrimidine biosynthesis
The pyrimidine end product, CTP, feedback to bind the R subunits, and inhibits catalytic activity of the C-trimers through a transmitted conformational change
In the presence of ATP ( a purine), the inhibition by CTP is prevented

54. Allosteric Enzyme ATCase
Active relaxed form
Carbamoyl
phosphate
Aspartate
Carbamoyl aspartate
COO-
CH2
HN-C-COO-
H H - O
H2N-C- =
COO-
CH2
N-C-COO-
H H - O
H2N-C-O-PO32- = +
ATCase
aspartate transcarbamoylase (ATCase)
ATP
Quaternary structure
CTP
Nucleic acid
metabolism
Feedback
inhibition
CCC
Catalytic subunits
CTP R R R
Regulatory subunits
CCC
Catalytic subunits
Inactive tense form
Juang RH (2004) BCbasics

55. Molecular structure of ATCase
Regulatory subunit
Catalytic subunit
A dodecamer C6R6 where C = catalytic, R = regulatory. ATCase assembles as a dimer of catalytic trimers plus three regulatory dimers

56. With the uninhibited enzyme, the two catalytic faces are sufficiently far apart to allow the substrate to enter between them and bind to the active site.
When CTP binds, it pulls the two faces together and effectively block access to the active site, inhibiting the enzyme.
substrate is red

57. Regulation of enzyme activity through covalent modification
reversible addition or removal of a chemical group which is covalently attached to protein (enzyme)
main examples are
• phosphorylation – dephosphorylation
• adenylation / uridylation / ADP- ribosylation
• methylation

58. Phosphorylation

59. Phosphorylation reaction
DG = -50 kJ/mol

60. The importance of Phosphorylation
The most common reversible covalent modification of proteins
Catalyzed by kinases
Adds two negative charges, altering electrostatic interactions
Phosphate groups also are effective at hydrogen bonding.
Phosphorylation may alter substrate binding and/or catalytic activity
Large negative ΔG for phosphorylation. About half of the -50 kJ/mol is conserved in the phosphorylated protein (the other half goes to making the reaction irreversible).

61. Main amino acid sites of phosphorylation (A) and adenylation (B)

62. Dephosphorylation
Phorphorylation is reversed by phosphatases

63. Phosphorylation-dephosphorylation

64. Example of reversibility of covalent modification

65. Adenylylation

66. Uridylylation

67. ADP-Ribosylation

68. Methylation

69. Regulation of enzyme activity by regulatory protein (subunit)
Active enzyme
Inactive enzyme
cAMP dependent protein kinase
R2C2 – Inactive R + 2C – Active
cAMP binds “R” ATP binds “C”

70. Regulation of enzyme activity by Proteolytic cleavage
Some enzymes are synthesized as proenzyme (zymogen), inactive form

71. Proteolysis also plays a regulatory role
Zymogens, or proenzymes, are inactive precursors of enzymes.
Cleavage in the chain causes irreversible conformational changes that expose the active site

72. Proteolysis of preproinsulin to active insulin
- Removal of leading peptide
- Specific folding stabilized by disulfide bonds
Connecting peptide removed, leaving complete two-chain insulin molecule

73. Regulation of carbohydrate metabolism
PART II
Regulation of carbohydrate metabolism

74. Carbohydrate, Lipid and Protein metabolisms are tightly integrated as Energy Metabolism
Main pathways are:
1. Glycolysis
2. Gluconeogenesis
3. Glycogen degradation and synthesis
4. Fatty acid degradation and synthesis
5. Citric acid cycle
6. Oxidative phosphorylation
7. Pentose phosphate pathway
8. Amino acid degradation and synthesis

75. Intrinsic vs Extrinsic Regulation
Intrinsic regulation
regulation by cell metabolites
Extrinsic regulation
regulation by external factors, i.e. hormones, stress, food, etc.

76. Intrinsic Regulation
Molecules such as NAD+, NADH, ATP, ADP, AMP are important allosteric effectors and intrinsic regulators of cellular metabolism.
●the concentrations of ATP-ADP-AMP mirror the energy charge of the cell
Energy charge (EC) = [ATP] + ½ [ADP]
[ATP] + [ADP] + [AMP]
high EC favors biosynthesis, low EC favors catabolism
●the NAD(P)/NAD(P)H ratio also regulates metabolism
low [NADH] promotes catabolism, high [NADH] = high ATP

77. If [ATP] is low, degradative pathways are stimulated.
If [ATP] is high, degradative pathways are inhibited.
Regulation of Synthesis and Degradation of glucose and glycogen depend on the energy state of the cell
Degradation
Synthesis

78. Extrinsic Regulation
Hormones are a higher order of regulation involving communication between cells, tissues, and the environment.
Hormones interact with receptors and set off a cascade of molecular events which:
• stimulate or repress the activity of key enzymes.
and / or
• stimulate or repress the transcription of specific genes .

79. Metabolic regulation by glucagon : an example of extrinsic regulation

80. Coordinated Regulation of Glycolysis and Gluconeogenesis

81. Regulation of glycolysis pathway

82. Regulation of Hexokinase (HK) : a conformational change
The active site pocket changes shape upon binding glucose
Only then can ATP transfer its phosphoryl group to the C6 carbon, yielding Glu-6-P + ADP
4 HK isozymes : HK I to III in muscle have Km 0.1 mM, active at low [glucose], use to consume glucose for energy production, allosterically inhibited by G-6-P
While HK IV (GK) in liver has high Km of 5-10 mM, active at high [glucose], suitable for maintaining blood glucose homeostasis, insensitive to G-6-P inhibition, reversibly inh by a regulatory protein in liver)

83. Vmax 100
Hexokinase
Glucokinase
Relative enzyme rate 50 Km
for HK Km
for GK
Blood Glucose
(mmol/L)
Physiological Significance of hexokinase and glucokinase kinetics as an example of comparative Km differences

84. Glucokinase kinetic Blood glucose level~5-10mM
GK is controlled by GK reg.protein, a competitive inhibitor of GK in the presence of F-6-P

85. Regulation of phosphofructokinase (PFK-1), a tetramer

86. Regulation and Energy signal of PFK-1

87. PFK-1 and F1,6-BP are reciprocally regulated

88. Gluconeogenesis 2 pyruvate + 2 NADH + 4 ATP + 2 GTP
Definition: the biosynthesis of glucose from non-carbohydrate molecules, primarily
pyruvate
lactate
some amino acid skeletons (glucogenic)
glycerol from fatty acids
2 pyruvate + 2 NADH + 4 ATP + 2 GTP
glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi
Gluconeogenesis expends 6 ~P bonds of ATP and GTP.

89. CONTROL:
• gluconeogenesis serves as an alternative source of glucose when supplies are low and is largely controlled by diet.
• high carbohydrate in meal reduce
gluconeogenesis and starvation increases.
• gluconeogenesis and glycolysis are controlled in reciprocal fashion. Two main points :
1. control PFK-1 and F1,6-BPase
2. control fate of pyruvate at PDH or Pyr carboxylase

90. Reciprocal regulation of glycolysis and gluconeogenesis

91. Fructose-2,6-bisphosphate (F2,6BP) is the
most important allosteric regulator of glycolysis and gluconeogenesis through its reciprocal effects on
fructose1,6-bisphosphatase and phosphofructokinase.
• not intermediate of both glycolysis and gluconeo-genesis, a product of PFK-2, under hormone control
• found in liver of all animals, some plants, and fungi, but not in bacteria

93. PFK-2/FBPase-2 is a bifunctional enzyme
Highly regulated to control the level of F2,6BP

95. Active forms of enzyme

96. Reciprocal control by F-2,6-BP

97. Control of PFK-1 by F-2,6-BP
F-2,6-BP allosterically activates PFK-1, promoting the relaxed state, even at relatively high [ATP].
Activity in the presence of F-2,6-BP is similar to that observed when [ATP] is low. Thus control by
F-2,6-BP, whose concentration fluctuates in response to external hormonal signals, supercedes control by local conditions (ATP concentration).

98. Pyruvate kinase (PK)
catalyzes substrate level phosphorylation, PEP Pyr
3 isozymes in vertebrates, all allosterically inh by ATP, AcCoA and long chain fatty acids
isoenzyme L (liver) – additionally regulated by glucagon via phosphorylation
isoenzyme M (muscle)
feedforward activated by F1,6BP, feedback inhibition by ATP

99. Regulation of PK

100. Regulation of Pyruvate kinase

101. Regulation of glycolysis and gluconeogenesis by control fate of pyruvate through PDH and PC
Two alternative fates for Pyruvate
AcCoA is an allosteric modulator to both enzymes

103. PDH catalyzes decarboxylation of pyruvate

104. PDH phosphorylation

105. PDH multienzyme complex
consists of 3 enzymes, E1 – Pyr DH tetramer, E2 – dihydro-lipoamide (DHLA) acetyl transferase, E3 – DHLA DH
involves 5 coenzymes / prosthetic group : CoASH, NAD+, TPP, Lipoate, FAD
TPP, Lipoate, FAD bind E1,E2,and E3,respectively, then pyruvate comes in, transfer acetyl group to E2-lipoyl, then CoASH comes to receive Ac. And released as AcCoA , finally NAD comes in to receive H+ from FADH2 bound at E3
all 3 enzymes are controlled by covelent modi. and allosteric reg. through 2 regulatory enzymes : PDH kinase and PDH phosphatase

106. Regulation of mammalian pyruvate dehydrogenase complex

107. Pyruvate Carboxylase
Mitochondrial enzyme, convert Pyr + HCO3- + ATP OAA + ADP + Pi, require biotin as cofactor
The 1st regulatory enzyme in gluconeogenesis, AcCoA is a positive regulator
OAA malate, shuttle to Cytoplasm OAA
PEP (reversible step)
MDH
MDH
PEP Carboxykinase

108. Coordinated Regulation of glycogen synthesis and breakdown in animals I. Glycogen phosphorylase is regulated allosterically and hormonally
Regulation of muscle GP by phosphorylation

109. Glycogen phosphorylase
allosteric effect is mediated by covalent modification

110. Glycogen phosphorylase (form a)
Phosphorylation at Ser14 in response to epinephrine (muscle)/glucagon (liver) destabilizes an N-terminal segment from an acidic to a basic environment
Allosterically activated by AMP (indicating ATP levels are low), noncovalently
Addition of glucose exposes Ser14–P to phosphatase, converting enzyme to the inactive b-form
yellow – P site
dark blue -allo site(AMP)
red - active site
light blue - cofactor (PLP)

111. Activity Regulation of Glycogen Phosphorylase
Covalent modification P A P A
GP kinase T P T
GP phosphatase 1 P
Non-covalent
ATP
Glc-6-P
Glucose
Caffeine
spontaneously A
Glucose
Caffeine
AMP
Garrett & Grisham (1999) Biochemistry (2e) p.679 P A P A A R R A

112. Cascade mechanism of epinephrine (muscle, liver) and glucagon (liver)

113. II. Glycogen Synthase is regulated by Phosphorylation/dephosphorylation

114. Overview of Regulation of Carbohydrate Metabolism in Liver

115. Carbohydrate Metabolism in liver vs in muscle

116. References
1. Lehninger Principles of Biochemistry, 5th Edition, 2008, D. L. Nelson and M.M. Cox, W.H.Freeman & Co., NY, Chapter 15, 6
2. Biochemistry,3rd ed.,2004, D. Voet and J.G.Voet, John Wiley & Sons, Inc.,USA., Chapter 10,16-18