1. Glycolysis Biochemistry by Reginald Garrett and Charles Grisham
Chapter 18
Glycolysis
Biochemistry by
Reginald Garrett and Charles Grisham

2. 18.1 – What Are the Essential Features of Glycolysis?
The Embden-Meyerhof (Warburg) Pathway: Glycolysis
Consists of two phases:
First phase converts glucose to two Glyceraldehyde-3-P
Energy investment phase
Consumes 2 molecules of ATP
Second phase produces two pyruvates
Energy generation phase
Produces 4 molecules of ATP
Products are 2 pyruvate, 2 ATP and 2 NADH
Essentially all cells carry out glycolysis
Ten reactions - same in all cells - but rates differ

3. Figure 18.1 The glycolytic pathway.

4. Figure Pyruvate produced in glycolysis can be utilized by cells in several ways. In animals, pyruvate is normally converted to acetyl-coenzyme A, which is then oxidized in the TCA cycle to produce CO2. When oxygen is limited, pyruvate can be converted to lactate. Alcoholic fermentation in yeast converts pyruvate to ethanol and CO2.

5. 18.2 – Why Are Coupled Reactions Important in Glycolysis?
Coupled reactions convert some, but not all, of the metabolic energy of glucose into ATP
The free energy change for the conversion of glucose to two lactates is
Glucose  2 lactate + 2H DG0’ = kJ/mol
The production of two molecules of ATP in glycolysis is an energy-required process
2 ADP + 2 Pi  2 ATP + 2 H2O DG0’ = 61 kJ/mol

6. Glycolysis couples these two reactions
Glucose  2 lactate + 2H DG0’ = kJ/mol
2 ADP + 2 Pi  2 ATP + 2 H2O DG0’ = 61 kJ/mol
Glucose + 2 ADP + 2 Pi  2 lactate + 2 ATP + 2H+ + 2 H2O
DG0’ = kJ/mol
More than enough free energy is available in the conversion of glucose into lactate to drive the synthesis of two molecules of ATP

7. 18.3 – What Are the Chemical Principles and Features of the First Phase of Glycolysis?

8. Under cellular condition DG = DGo’ + RT ln ( [G-6-P][ADP] / [Glu][ATP] )

9. Phase 1 Phosphorylation (Hexokinase)
Isomerization (Phosphoglucoisomerase)
Phosphorylation (Phosphofructokinase)
Cleavage (Aldolase)
Isomerization (Triose phosphate isomerase)

10. Phosphorylation of glucose
Reaction 1: Hexokinase
Phosphorylation of glucose
Hexokinase (or glucokinase in liver)
This is a priming reaction - ATP is consumed here in order to get more later
ATP makes the phosphorylation of glucose spontaneous
Mg2+ is required
Mg2+

11. The cellular advantages of phosphorylating glucose
Phosphorylation keeps the substrate in the cell
Keeps the intracellular concentration of glucose low, favoring diffusion of glucose into the cell
Makes it an important site for regulation
Figure 18.4 Glucose-6-P cannot cross the plasma membrane.

12. Hexokinase 1st step in glycolysis; G large, negative
Km for glucose is 0.1 mM; cell has 4 mM glucose, so hexokinase is normally active
Hexokinase is regulated --allosterically inhibited by (product) glucose-6-P -- but is not the most important site of regulation of glycolysis—Why? (Figure 18.5)
Can phosphorylate a variety of hexose sugars, including glucose, mannose, and fructose

13. Figure 18.5 Glucose-6-phosphate is the branch point for several metabolic pathways.

14. Glucokinase The isozymes of hexokinase
Hexokinase I in brain
Hexokinase I (75%, 0.03mM) and II (25%, 0.3mM) in muscle
Glucokinase in liver and pancreas
Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in glucose
Is not product inhibited
Is an inducible enzyme by insulin

15. Induced fit model (fig 13.24)
Figure The (a) open and (b) closed states of yeast hexokinase
Figure 18.7 (a) Mammalian hexokinase I

16. Reaction 2: Phosphoglucoisomerase
Glucose-6-P (aldose) to Fructose-6-P (ketose)
Why does this reaction occur
next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH
isomerization activates C-3 for cleavage in aldolase reaction
Phosphoglucose isomerase or glucose phosphate isomerase
Ene-diol intermediate in this reaction

17. Phosphoglucoisomerase, with fructose-6-P (blue) bound.

18. Figure 18.8 The phosphoglucoisomerase mechanism involves opening of the pyranose ring (step 1), proton abstraction leading to enediol formation (step 2), and proton addition to the double bond, followed by ring closure (step 3)

19. Reaction 3: Phosphofructokinase
PFK is the committed step in glycolysis
The second priming reaction of glycolysis
Committed step and large, negative DG -- means PFK is highly regulated
Fructose-6-P + Pi → Fructose-1,6-bisP DGo’= 16.3 kJ/mol

20. Regulation of Phosphofructokinase
ATP also is a allosteric inhibitor
Has two distinct binding sites for ATP (A high-affinity substrate site and a low-affinity regulatory site)
AMP reverses the inhibition due to ATP
Raise dramatically when ATP decrease
Citrate is also an allosteric inhibitor
Fructose-2,6-bisphosphate is allosteric activator
PFK increases activity when energy status is low
PFK decreases activity when energy status is high

21. Figure At high ATP, phosphofructokinase (PFK) behaves cooperatively and the activity plot is sigmoid.

22. Figure Fructose-2,6-bisphosphate activates phosphofructokinase, increasing the affinity of the enzyme for fructose-6-phosphate and restoring the hyperbolic dependence of enzyme activity on substrate concentration.

23. Figure 18.11 Fructose-2,6-bisphosphate decreases the inhibition of phosphofructokinase due to ATP.

24. Reaction 4: Fructose Bisphosphate Aldolase
C6 is cleaved to 2 C3s (DHAP, Gly-3-P)
Fructose bisphosphate aldolase cleaves fructose-1,6-bisphosphate between the C-3 and C-4 carbons to yield two triose phosphates
Dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G-3-P)

25. The aldolase reaction is unfavorable as written at standard state
The aldolase reaction is unfavorable as written at standard state. The cellular ΔG, however, is close to zero.
The aldolase reaction in glycolysis is merely the reverse of the aldol condensation well known to organic chemists.

26. Animal aldolases are Class I aldolases
Class I aldolases form covalent Schiff base intermediate between substrate and active site lysine

27. Class II aldolase are produced mainly in bacteria and fungi

28. Reaction 5: Triose Phosphate Isomerase
Only G-3-P goes directly into the second phase, DHAP must be converted to G-3-P
Triose phosphate isomerase
An ene-diol mechanism
Active site Glu acts as general base
is a near-perfect enzyme (Table 13.5)

29. Figure 18. 13 A reaction mechanism for triose phosphate isomerase
Figure A reaction mechanism for triose phosphate isomerase. In the yeast enzyme, the catalytic residue is Glu165.

30. 18.4 – What Are the Chemical Principles and Features of the Second Phase of Glycolysis?
Metabolic energy produces 4 ATP
Net ATP yield for glycolysis is two ATP
Second phase involves two very high energy phosphate intermediates
1,3 BPG
Phosphoenolpyruvate
Substrate-level phosphorylation

32. Phase 2
Oxidation and Phosphorylation (Glyceraldehyde-3-P Dehydrogenase)
Substrate-level Phosphorylation (phosphoglycerate kinase)
Isomerization (Phosphoglycerate isomerase)
Dehydration (Enolase)
Substrate-level Phosphorylation (pyruvate kinase)

33. Reaction 6: Glyceraldehyde-3-Phosphate Dehydrogenase
G-3-P is oxidized to 1,3-BPG
Energy yield from converting an aldehyde to a carboxylic acid is used to make 1,3-BPG and NADH
Oxidation (aldehyde to carboxylic acid) and phosphorylation

34. G3PDH (or GAPDH)
Mechanism involves covalent catalysis and a nicotinamide coenzyme

35. Reaction 7: Phosphoglycerate Kinase
ATP synthesis from a high-energy phosphate
This is referred to as "substrate-level phosphorylation"
Coupled reactions; 6th and 7th reactions
Glyceraldehyde-3-P + ADP + Pi + NAD+ →
3-phosphoglycerate + ATP + NADH + H+ DGo’= kJ/mol

36. ATP is synthesized by three major routes:
Substrate-level phosphorylation (Glycolysis, Citric acid cycle)
Oxidative phosphorylation (Driven by electron transport)
Photophosphorylation (Photosynthesis)

37. 2,3-BPG (for hemoglobin) is made by circumventing the PGK reaction
Bisphosphoglycerate mutase
Erythrocytes contain 4-5 mM 2,3-BPG
Figure The mutase that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglycerate.

38. Reaction 8: Phosphoglycerate Mutase
Repositions the phosphate
Mutase: catalyzes migration of a functional group within a substrate

39. Phosphoenzyme intermediates A bit of 2,3-BPG is required as a cofactor
The catalytic His183 at the active site of E. coli phosphoglycerate mutase

40. Reaction 9: Enolase The formation of PEP from 2-PG Dehydration
Make a high-energy phosphate in preparation for ATP synthesis in step 10 of glycolysis

41. Reaction 10: Pyruvate Kinase
The pyruvate kinase reaction converts PEP to pyruvate, driving synthesis of ATP.
Substrate-level phosphorylation
Another key control point for glycolysis
Enol-keto tautomer .

42. Figure The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer, followed by an enol-keto tautomerization. The tautomerization is spontaneous and accounts for much of the free energy change for PEP hydrolysis.

43. Large, negative G -- regulation
Allosteric regulation
Activated by AMP, F-1,6-bisP
Inhibited by ATP ,acetyl-CoA, and alanine
Liver pyruvate kinase is regulated by covalent modification
Responsive to hormonally-regulated phosphorylation in the liver (glucagon)
The phosphorylated form of the enzyme is more strongly inhibited by ATP and alanine.
Has a higher Km for PEP

44. 18.5 – What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis?
Aerobic or anaerobic?
NADH must be recycled to NAD+
If O2 is available, NADH is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation (chapter 20)
In anaerobic conditions, NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis

45. Figure (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD+ consumed in the glyceraldehyde-3-P dehydrogenase reaction. (b) In oxygen-depleted muscle, NAD+ is regenerated in the lactate dehydrogenase reaction.

46. Pyruvate also has two possible fates:
aerobic: into citric acid cycle (chapter 19) where it is oxidized to CO2 with the production of additional NADH (and FADH2)
anaerobic: (fermentation)
In yeast: reduced to ethanol
Pyruvate decarboxylase (TPP)
Alcohol dehydrogenase (Reoxidized NADH to NAD+)
In animals: reduced to lactate
Lactate dehydrogenase (Reoxidized NADH to NAD+)

47. 18.6 – How Do Cells Regulate Glycolysis?
The elegant evidence of regulation (See Figure 18.22)
Standard state G values are variously positive and negative
G in cells is revealing:
Most values near zero (reactions 2 and 4-9)
3 of 10 Reactions have large, negative G
Large negative G Reactions are sites of regulation
Hexokinase
Phosphofructokinase
Pyruvate kinase

49. Overview of the regulation of glycolysis.

50. 18.7 – Are Substrates Other Than Glucose Used in Glycolysis?
Sugars other than glucose can be glycolytic substrates
Fructose and mannose are routed into glycolysis by fairly conventional means.

51. Fructose In liver In kidney and muscle Fructokinase
Fructose + ATP  fructose-1-phosphate + ADP + H+
Fructose-1-phosphate aldolase
fructose-1-phosphate  glyceraldehyde + DHAP
Triose kinase
glyceraldehyde  glyceraldehyde-3-phosphate
In kidney and muscle
Hexokinase
Fructose + ATP  fructose-6-phosphate + ADP + H+

52. Mannose
Hexokinase
mannose + ATP  mannose-6-phosphate + ADP + H+
Phosphomannoisomerase
mannose-6-phosphate  fructose-6-phosphate
Galactose is more interesting - the Leloir pathway "converts" galactose to glucose
Galactokinase
Galactose + ATP  galactose-1-phosphate + ADP + H+
Galactose-1-phosphate uridylyltransferase
Phosphoglucomutase
UDP-galactose-4-epimerase

53. Figure 18.24 Galactose metabolism via the Leloir pathway.

54. Figure The galactose-1-phosphate uridylyltransferase reaction involves a “ping-pong” kinetic mechanism.

55. Galactosemia Defects in galactose-1-P uridylyltransferase
Galactose accumulate causes cataracts and permanent neurological disorders
In adults, UDP-glucose pyrophosphorylase also works with galactose-1-P, reducing galactose toxicity

56. Lactose Intolerance
The absence of the enzyme lactase (b-galactosidase)
Diarrhea and discomfort

57. Glycerol can also enter glycolysis
Glycerol is produced by the decomposition of triacylglycerols (chapter 23)
Converted to glycerol-3-phosphate by the action of glycerol kinase
Then oxidized to DHAP by the action of glycerol phosphate dehydrogenase
NAD+ as the required coenzyme

58. 18.8 How Do Cells Respond to Hypoxic Stress?
Glycolysis is an anaerobic pathway—it does not require oxygen
The TCA (tricarboxylic acid) cycle is aerobic. When oxygen is abundant, cells prefer to combine these pathways in aerobic metabolism
When oxygen is limiting, cells adapt to carry out more glycolysis

59. Hypoxia (oxygen limitation) causes changes in gene expression that increases
Angiogenesis (the growth of new blood vessels)
Synthesis of red blood cells
Levels of some glycolytic enzymes (a high rate of glycolysis)
Hypoxic stress
A trigger for this is a DNA binding protein called hypoxia inducible factor (HIF)
HIF is regulated at high oxygen levels by hydroxylase factor-inhibiting HIF (FIH-1)

60. Hypoxia inducible factor (HIF)
A heterodimer consists of two subunits:
A constitutive, stable nuclear b subunit HIF-1β
An inducible, unstable hypoxia-responsive HIF-α subunit
Bind to the hypoxia responsive element (HRE) of hypoxia-inducible genes—Activating transcription of these genes
HIF-α regulation is a multistep process
Gene splicing
Acetylation (Inhibited)
Hydroxylation (Inhibited)
Phosphorylation (activated)

61. Under normal oxygen levels, HIF-a are synthesized but quickly degraded
When oxygen is plentiful, HIF-1a is hydroxylated by the prolyl hydroxylases (PHDs)
These hydroxylation ensure its binding to ubiquitin E3 ligase, which leads to rapid proteolysis
HIF-1a binding to the ubiquitin E3 ligase is also promoted by acetylation by the ARD1 acetyltransferase
FIH-1 (hydroxylase factor-inhibiting HIF-a) hydroxylates HIF-a at Asn803
PHDs and FIH-1 both are oxygen-dependent

62. Figure 18.28