1. The synthesis of glucose from noncarbohydrate precursors
GLUCONEOGENESIS
The synthesis of glucose from noncarbohydrate precursors
Mainly in the liver

2. IMPORTANT PRINCIPLES OF BIOSYNTHESIS
The synthesis pathway is usually different from the degradation pathway.
The two opposing pathways may share many reversible reactions.
There is always at least one unique enzymatic step to each pathway.
BECAUSE
If this wasn’t the case, then the flow of carbon through the two pathways would be dictated by the mass action and not the cellular changing needs for energy, precursors or macromolecules.

3. IMPORTANT PRINCIPLES OF BIOSYNTHESIS
Corresponding anabolic and catabolic pathways are controlled by different regulatory enzymes in a reciprocal manner.
If one pathway is stimulated, then the opposite is inhibited.
Biosynthetic pathways are usually regulated at their initial steps.
BECAUSE
To prevent wasting precursors to make unneeded intermediates.

4. IMPORTANT PRINCIPLES OF BIOSYNTHESIS
The energy-requiring biosynthetic processes are coupled to the energy-yielding hydrolysis of ATP
The overall process is essentially irreversible in vivo.
The total amount of energy from ATP (and NAD(P)H) is larger than the minimum energy needed to convert the precursor into the biosynthetic product.
THUS
The resulting large negative free energy for the overall process will assure that it will take place even when the concentrations of the precursors are relatively low.

5. Noncarbohydrate precursors of glucose:
Triglycerols
glycerol
Fatty acids
Dietary & muscle
proteins
Amino acids

6. Main sites of gluconeogenesis:
Major site: Liver.
Minor site: Kidney.
Very little:
Brain.
Muscle (skeletal and heart).
gluconeogenesis in the liver and kidney helps to maintain the glucose level in the blood so that brain and muscle can extract sufficient glucose from it to meet their metabolic demands.

7. Gluconeogenesis Is Not a Reversal of Glycolysis:
Seven steps are shared by glycolysis and gluconeogenesis.
However, three essentially irreversible steps in glycolysis shift the equilibrium far in the side of glycolysis.
Most of the decrease in free energy in glycolysis takes place in these steps.

9. In gluconeogenesis the three reactions are bypassed by a set of separate enzymes.
Phosphoenolpyruvate is formed from pyruvate:
Fructose 6-phosphate is formed from fructose 1,6-bisphosphate:
Glucose is formed by hydrolysis of glucose 6-phosphate:

10. PYRUVATE PHOSPHOENOLPYROVATEB

11. PYROVATE CARBOXYLASE
Mitochondrial enzyme

12. CARBOXYLATION OF PYROVATE:1
HCO3-, the aqueous form of CO2 is activated to carboxyphosphate. 2
it is subsequently activated by binding to the N-1 atom of the biotin ring to form the carboxybiotin-enzyme intermediate. The DG°´ for its cleavage is -20 kJ mol-1. 3
The activated carboxyl group is then transferred from carboxybiotin to pyruvate to form oxaloacetate.

14. The long, flexible link between biotin and the enzyme enables this prosthetic group to rotate from one active site of the enzyme (the ATP-bicarbonate site) to the other (the pyruvate site).
To be carboxylated, biotin needs the enzyme to be allosterically activated by Acetyl CoA.

15. DECARBOXYLATION AND PHOSPHORYLATION OF OXALOACETATE:
Oxaloacetate, is reduced to malate inside the mitochondrion for transport to the cytosol.
The reduction is accomplished by an NADH-linked malate dehydrogenase.
When malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD+-linked malate dehydrogenase in the cytosol.

16. Oxaloacetate is simultaneously decarboxylated and phosphorylated by phosphoenolpyruvate carboxykinase in the cytosol.
The CO2 that was added to pyruvate by pyruvate carboxylase comes off in this step.
The formation of the unstable enol is driven by decarboxylation, and trapped by the addition of a phosphate to carbon 2 from GTP.

17. PEP + H+ NAD+ NAD+ H++ Pyruvate Oxaloacetate CO2 Malate Malate
Cytosolic PEP CARBOXYKINASE
Oxaloacetate
PEP
Cytosolic Malate DEHYDROGENASE
NADH
+ H+
CO2
NAD+
Malate
Malate
NAD+
NADH
H++
Oxaloacetate
Pyruvate CARBOXYLASE
CO2
Pyruvate
Pyruvate

18. This is accomplished by transporting Malate outside the mitochondria.
This pathway predominates when pyruvate or alanine is the glucogenic precursor.
The carboxylation-decarboxylation represents a way of activating pyruvate.
the decarboxylation of Oxaloacetate facilitates PEP formation.
The cytosolic NADH is consumed by other gluconeogenesis reactions, and has to be regenerated in order to proceed with the process.
This is accomplished by transporting Malate outside the mitochondria.

19. PEP PEP + H+ NAD+ NAD+ H++ + H+ NAD+ Oxaloacetate CO2 Malate Malate
Cytosolic PEP CARBOXYKINASE
Oxaloacetate
PEP
Cytosolic Malate DEHYDROGENASE
NADH
+ H+
CO2
NAD+
Malate
PEP
Malate
Mitochondrial PEP CARBOXYKINASE
NAD+
CO2
H++
NADH
Oxaloacetate
Oxaloacetate
Pyruvate CARBOXYLASE
Pyruvate CARBOXYLASE
CO2
CO2
Pyruvate
Pyruvate
Pyruvate
NADH
+ H+
Pyruvate
NAD+
Lactate DEHYDROGENASE
Lactate

20. This pathway predominates when lactate is the precursor.
The conversion of lactate to pyruvate in the hepatocyte cytosole yields NADH.
Thus no Malate transport is needed any more for this purpose.
The mitochondrial and cytosolic PEP CARBOXYKINASE enzymes are encoded by separate nuclear genes. (two different enzymes catalyzing the same reaction in different localizations)

21. FRUCTOSE 1,6-BISPHOSPHATE FRUCTOSE 6-PHOSPHATE
The enzyme responsible for this step is fructose 1,6-bisphosphatase.
Like its glycolytic counterpart, it is an allosteric enzyme that participates in the regulation of gluconeogenesis.

22. GLUCOSE 6-PHOSPHATE GLUCOSE
This hydrolytic reaction is catalyzed by glucose-6-phosphatase in the Endoplasmic reticulum of the hepatocytes..
In most tissues, free glucose is not generated;
the glucose 6-phosphate is processed in some other fashion, notably to form glycogen.
Unlike free glucose, glucose 6-phosphate cannot diffuse out of the cell.

23. To keep glucose inside the cell, the generation of free glucose is controlled in two ways:
The enzyme responsible for the conversion of glucose 6-phosphate into glucose, glucose 6-phosphatase, is regulated.
The enzyme is present only in tissues whose metabolic duty is to maintain blood-glucose homeostasis tissues that release glucose into the blood (the liver and to a lesser extent the kidney).

24. Several endoplasmic reticulum (ER) proteins play a role in the generation of glucose from glucose 6-phosphate.
T1 transports glucose 6-phosphate into the lumen of the ER.
T2 and T3 transport Pi and glucose, respectively, back into the cytosol.
Glucose 6-phosphatase is stabilized by a Ca2+-binding protein (SP).

33. REGULATION OF CARBOHYDRATE METABOLISM

34. The rate of conversion of glucose into pyruvate is regulated to meet two major cellular needs:
The production of ATP, generated by the degradation of glucose.
The provision of building blocks for synthetic reactions, such as the formation of fatty acids.

35. It is all in the enzymes
Enzymes can enhance the rates of metabolic (or other) reactions by many orders of magnitude.
A rate enhancement of 1017 means that what would occur in 1 second with an enzyme’s help, would otherwise require 31,710,000,000 years to take place.
So essentially without enzymes such reactions don’t take place.
Thus, regulation of enzymatic activity is in a sense, regulation of metabolism, or any other cellular process.

36. Regulation and control of enzyme activity
Substrate level control.
Allosteric effectors
Covalent modification
Enzyme concentration: increased synthesis
Enzyme concentration: generation of active enzyme by processing
Substrate cycles

37. Substrate level control
Since most often [S] > Km, the change in substrate concentration does not change the reaction rate appreciably.
Thus, controlling a metabolic flux is not normally achieved by varying substrate concentrations.

38. Allosteric effectors Noncovalently bind and regulate the enzyme.
The effector may be stimulatory or inhibitory.
The substrate and effector usually occupy different specific binding sites.

39. Allosteric enzymes kinetics:
Sigmoid kinetic behavior is seen.
K0.5 represents the substrate concentration at which the enzyme velocity is half Vmax.
(-) and (+) respectively indicate inhibitory and stimulatory effectors.

40. Covalent modification

41. Enzyme concentration: increased synthesis

42. Enzyme concentration: generation of active enzyme by processing

43. Regulation of the flux through multistep pathways occurs at steps that are enzyme limited:
RATE-LIMITING STEP: the rate of at least one reaction in every metabolic pathway depends on the activity of the enzyme (ENZYME-LIMITED), and is not limited to the substrate availability.

44. Any enzyme that catalyzes the 1st step in a pathway is a potential control point since it shows “commitment” to the pathway.
Phosphofructokinase is the obvious point in glycolysis.
Any enzyme that is working slowly (small Vmax) is obviously a bottle-neck in the reaction.
Therefore activation of a slow enzyme can increase the flux of the entire pathway.
In heart muscle glycolysis the slowest enzymes are:
Hexokinase.
Phosphofructokinase.
Aldolase.
Enolase.

45. Their activities are regulated by:
Irreversible reactions in glycolysis (rate-limiting) are potential sites of control.
Hexokinase
Phosphofructokinase
pyruvate kinase.
Their activities are regulated by:
the reversible binding of allosteric effectors
by covalent modification.
the amounts of these important enzymes are varied by the regulation of transcription to meet changing metabolic needs.

46. Gluconeogenesis and Glycolysis Are Reciprocally Regulated
The amounts and activities of the distinctive enzymes of each pathway are controlled so that both pathways are not highly active at the same time.
The interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate is stringently controlled.
Phosphofructokinase and fructose 1,6-bisphosphatase are reciprocally controlled by fructose 2,6-bisphosphate in the liver

48. ALLOSTERIC REGULATORS OF PFK-1 and FBPase-1

49. PHOSPHOFRUCTOKINASE: The most important control element in the mammalian glycolytic pathway.
PFK in the liver is a tetramer of 4 identical subunits.
The allosteric effectors binding site is distinct from the catalytic site.

50. ATP allosterically inhibit the enzyme:
High concentrations of ATP converts the hyperbolic binding curve of F6-P to sigmoidal one.
AMP reverses the inhibitory effect of ATP
The activity of the enzyme increases when the ATP/AMP ratio is lowered
glycolysis is stimulated
as the energy charge falls

51. WHY is AMP rather than ADP the positive regulator of PFK-1?
When ATP is utilized rapidly, the enzyme Adenylate Kinase forms ATP and AMP from ADP:
AMP becomes the signal for low energy charge.
ADP + ADP ATP + AMP

52. Citrate inhibit the PFK-1 enzyme
A high level of citrate means that biosynthetic precursors are abundant and additional glucose should not be degraded for this purpose.
Citrate inhibits PFK-1 by enhancing the inhibitory effect of ATP.

53. F2,6-BP allosterically activates PFK-1 and inhibits FBPase:
Phosphofructokinase and fructose 1,6-bisphosphatase are reciprocally controlled by fructose 2,6-bisphosphate in the liver.

54. The sigmoidal dependence of velocity on substrate concentration becomes hyperbolic.
ATP acting as substrate initially stimulates the reaction and afterward acts as allosteric inhibitor
The inhibitory effect of ATP is reverse by F2.6-BP

55. How is the concentration of F 2,6-BP appropriately controlled?
F2,6-BP is formed in a reaction catalyzed by Phosphofructokinase-2 (PFK-2)
It is hydrolyzed to F6-P by Fructose Bisphosphatase-2 (FBPase-2)
Both PFK-2 and FBPase-2 are part of the same 55Kd polypeptide chain.
The bifunctional enzyme
Exists in 5 isozymic forms.
L-isoform in liver.
M-isoform in muscle.

56. The L-isoform help to maintain blood-glucose homeostasis:
Feedforward stimulation

57. Hexokinase:
GLUCOKINASE
ATP
ADP
In LIVER
HEXOKINASE
glucose 6-phosphate
accomulates
Synthesis of glycogen and
fatty acids.
PHOSPHOGLUCOSE
ISOMERASE
Glucokinase has ~ 50-fold lower affinity for glucose than does hexokinase.
The low affinity in the liver gives the brain and muscles first call on glucose when its supply is limited, whereas it ensures that glucose will not be wasted when it is abundant.
Fructose 6-phosphate
accomulates
PHOSPFOFRUCTOKINASE

58. Why is phosphofructokinase rather than hexokinase the pacemaker of glycolysis?
Glucose 6-phosphate is not solely a glycolytic intermediate.
It can also be converted into glycogen or it can be oxidized by the pentose phosphate pathway to form NADPH.
The first irreversible reaction unique to the glycolytic pathway, the committed step, is the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate.

59. The interconversion of PEP and pyruvate is precisely regulated.
gluconeogenesis is favored when the cell is rich in biosynthetic precursors and ATP.

60. Pyruvate kinase L-isoform
Pyruvate kinase is regulated by allosteric effectors and covalent modification.
These hormone-triggered phosphorylations, prevent the liver from consuming glucose when it is more urgently needed by brain and muscle
PHOSPHATASE

61. GLUCOSE TRANSPORTRS Name Tissue location Km Comments GLUT1
All mammalian tissues
1 mM
Basal glucose uptake
GLUT2
Liver and pancreatic b cells
15-20 mM
In the pancreas, plays a role in regulation of insulin secretion
In the liver, removes excess glucose from the blood
GLUT3
GLUT4
Muscle and fat cells
5 mM
Amount in muscle plasma membrane increases by insulin and with endurance training
GLUT5
Small intestine
    
Primarily a fructose transporter

63. HORMONS control the amount and activities of essential enzymes
Hormones affect gene expression primarily by:
changing the rate of transcription
regulating the degradation of mRNA.
Transcriptional control in eukaryotes is much slower than allosteric control;
it takes hours or days in contrast with seconds to minutes.

64. Insulin Rises subsequent to eating and stimulates the expression of:
Phosphofructokinase
pyruvate kinase
PFK-2/FBPase-2

65. Glucagon Rises during starvation: inhibits the expression of:
Phosphofructokinase
pyruvate kinase
PFK-2/FBPase-2.
stimulates instead the production of two key gluconeogenic enzymes:
phosphoenolpyruvate carboxykinase
fructose 1,6-bisphosphatase

66. Hormones work at the promoter level
The PEP-Carboxykinase promoter approximately 500 bp in length
Contains regulatory sequences (response elements) that mediate the action of several hormones:
IRE: insulin response element
GRE: glucocorticoid response element
TRE: thyroid hormone response element
CREI and CREII: cAMP response elements.

67. Substrate Cycles:
Both reactions are not simultaneously fully active in most cells, because of reciprocal allosteric controls.
However, some F6-P is phosphorylated to F1,6-BP in gluconeogenesis.
This cycling was regarded as an imperfection in metabolic control, and so substrate cycles have sometimes been called futile cycles.
F 6-P
F 1,6-BP
H2O Pi
F1,6-BPase
ATP
ADP
PFK-1

68. Substrate cycles are biologically important:
Substrate cycles amplify metabolic signals:
This amplification is made possible by the rapid hydrolysis of ATP.
If an allosteric effector reciprocally increases A to B and decreases B to A by 20% each
Then a 20% change in the rates of the opposing reactions has led to a 480% (=100x48/10) increase in the net flux.
generation of heat produced by the hydrolysis of ATP.

69. Lactate produced by active skeletal muscle and erythrocytes is a source of energy for other organs.
The only purpose of the reduction of pyruvate to lactate is to regenerate NAD+ so that glycolysis can proceed in active skeletal muscle and erythrocytes.
lactate is a dead end in metabolism.
It must be converted back into pyruvate before it can be metabolized.

70. The Cori cycle
Lactate in the liver is oxidized to pyruvate, a reaction favored by the low NADH/NAD+ ratio in the cytosol of liver cells.
Pyruvate in the liver is converted into glucose by the gluconeogenic pathway.
Glucose then enters the blood and is taken up by skeletal muscle.
Both pyrovate and lactate diffuse out of active skeletal muscle into the blood and are carried to the liver.
Much more lactate than pyruvate is transported out because the high NADH/NAD+ ratio in contracting skeletal muscle favors the conversion of pyruvate into lactate.

71. The interplay between glycolysis and gluconeogenesis

72. Lactate dehydrogenase isozymes
Tetramer of 2 kinds of 35kd subunits (H, M).
H mainly in heart
M mainly in skeletal muscle and liver
5 types of tetramers:
Type Composition Affinity to substrate Reaction 1 H4
H3M1
H2M2
H1M3 M4
High
Low
Lactate pyrovate 2 3 4 5
Lactate pyrovate

73. Hypoxia inducible factor (HIF-1) Increased vascularization
CANCER AND GLYCOLYSIS
Soled tumors grow
hypoxia
Hypoxia inducible factor (HIF-1)
Increased new tumors growth by inducing the expression of signal molecules like vascular endothlial growth factor VEGF
High expression of:
glycolytic enzymes
and GLUT1 and GLUT2
Increased tumor aggressiveness and poor prognosis
Increased vascularization

74. Proteins in glucose metabolism encoded by genes regulated by HIF-1:
GLUT1GLUT3
Hexokinase
Phosphofructokinase
Aldolase
Glyceraldehyde 3-phosphate dehydrogenase
Phosphoglycerate kinase
Enolase
Pyruvate kinase
Lactate dehydrogenase