5/30/13 version
Dr. Brad Chazotte
213 Maddox Hall chazotte@campbell.edu
Web Site: http://www.campbell.edu/faculty/chazotte
Original material only ©2000-14 B. Chazotte
Pharm. 304
Biochemistry
Fall 2014

•Learn the enzymes and sequence of reactions in glycolysis
•Develop an understanding of the chemical “logic” of the glycolysis pathway
•Understand the basis and need for redox balance in glycolysis
•Learn and understand the control(s) and control points of the glycolysis pathway.
•Learn where products of glycolysis can go.
•Be aware that other sugars can enter the glycolysis pathway

An Energy Conversion Pathway Used by Many Organisms
•Almost a universal central pathway for glucose catabolism
•The chemistry of these reactions has been completely conserved.
•Glycolysis differs among species only in its regulation and in the metabolic fate of the pyruvate generated.
•In eukaryotic cells glycolysis takes place in the cell cytosol.

The Glycolysis Pathway
[Embden-Meyerhof Pathway]
Glycolysis is the sequence of reactions that metabolizes one molecule of glucose to two molecules of pyruvate with the concomitant net production of two molecules of ATP
Glycolysis is an anaerobic process, i.e., it does not require oxygen
Voet, Voet & Pratt 2013 Fig 15.1

Overall Reaction of Glycolysis
Glucose + 2NAD + + 2ADP + 2P i
2 pyruvate + 2 NADH + 2H + + 2ATP + 2H
2
O
Conversion of glucose into pyruvate:

G
1

= -146 kJ mol -1
Glucose + 2NAD + 2 pyruvate + 2 NADH + 2H +
Formation of ATP from ADP and P i

G
2

= 2 (30.5)= 61 kJ mol -1
2ADP + 2P i
2ATP + 2H
2
O

G s

=

G
1

+

G
1

= -146 kJ mol -1 + 61 kJ mol -1 = -85 kJ mol -1

There are three major stages of glycolysis defined
(some texts define two):
•Trapping and destabilization of glucose (2 ATP used)
•Cleavage of 6-carbon fructose to two interconvertible
3-carbon molecules (4 ATP produced)
•Generation of ATP

Examples of Glucose Metabolic Fates
Major Glucose Utilization Pathways in Cells of Higher Plants and Animals
Catabolism via Pyruvate
Pyruvate
O O
-
CH
3
Voet, Voet & Pratt 2013 Fig 15.16
C C O
Lehninger 2000 Fig 15.1

FERMENTATION
Definition: A general term for the anaerobic degradation of glucose or other organic nutrients to obtain energy conserved in the form of ATP.
Disadvantage:
Advantage:
Fermentations produce less energy than complete combustion with oxygen
Does not require oxygen. Gives an organism a wider choice of habitats.
TWO EXAMPLES OF FERMENTATION:
Alcohol Fermentation: e.g. the conversion of pyruvate from glycolysis to ethanol in yeast CH
3
-CH
2
OH
Lactic Acid Fermentation: e.g. the conversion of pyruvate from glycolysis to lactic acid in skeletal muscle. CH
3
-CHOH-COO -

Reactions of Glycolysis
Berg, Tymoczko & Stryer, 2012 Table. 16.1

Schematic of the
Glycolysis
Pathway
Horton 2-stage
Hexose stage
Triose stage
1. Trap and destabilize
2. Cleave 6-C into two 3-C molecules
3. Generate ATP
Berg, Tymoczko & Stryer, 2002 Fig. 16.3

Stage 1 of Glycolysis Detail
Berg, Tymoczko & Stryer, 2002 Fig. 16.X

Conversion of Glucose by Hexokinase carbon numbering
Hexokinase present in all cells of all organisms
Kinases are enzymes that catalyze the transfer of a phosphoryl group from ATP to an acceptor mechanism
Reaction Purposes:
1. Traps glucose in the cell due to the negative charges on the phosphoryl groups which are ionized at pH 7.
Precludes diffusion through the plasma membrane.
2. The attachment of the phosphoryl group renders glucose a less stable molecule and more amenable to further metabolic action .

G

= -16.7 kJ/mol
Glycolysis Step 1
Lehninger 2000 Fig 15.1
Horton, 2002 Fig 11.3

Yeast Hexokinase
Two lobes move towards each other as much as 8 Å when glucose is bound
Resulting cavity creates a much more nonpolar environment around the glucose molecule which favors the donation of the
ATP’s terminal phosphate
Berg, Tymoczko & Stryer, 2012 Fig. 16.3
Voet, Voet & Pratt , 2008 Fig. 15.2

Isomerization of Glucose-6-P to Fructose-6-P
Glycolysis Step 2

G

=1.7 kJ/mol
Berg, Tymoczko & Stryer, 2012 Chap 16 p. 457

Enzyme active site
Lys?
Glu?
Glycolysis Step 2
Voet, Voet & Pratt 20012 Fig. 15.3

Phosphorylation of Fructose 6-P
Glycolysis Step 3

G

= -14.2 kJ/mol
Berg, Tymoczko & Stryer, 2012 Chap 16

Stage 2 of Glycolysis
Berg, Tymoczko & Stryer, 2002 chap 16.
Berg, Tymoczko & Stryer, 2002 Chap. 16

Cleavage of Fructose 1,6-biphosphate by Aldolase
Glycolysis Step 4

G

=23.8 kJ/mol
Berg, Tymoczko & Stryer, 2012 chap 16 p. 458

Glycolysis Step 4
Voet, Voet & Pratt 2013 15 p. 478

Glycolysis
Voet, Voet & Pratt 2013 Fig. 15.4

Aldolase Mechanism
The cleavage by aldolase of
F1,6BP stabilizes the enolate intermediate via increased electron delocalization.
Voet, Voet & Pratt 2013 Fig. 15.5

Stage 2 of Glycolysis
End of “stage I ” in Voet, Voet & Pratt
Berg, Tymoczko & Stryer, 2002 Chap 16.

Isomerization of Dihdroxyacetone phosphate
Glycolysis Step 5

G

=7.5kJ/mol
Berg, Tymoczko & Stryer, 2002 Fig. 16.3

Isomerization of DHAP with Carbon #s
Lehninger 2000 Fig 15.4

Cunningham 1978, p343

x
Glycolysis Step 5
Voet & Voet Biochemistry 1995 Fig.16.10

Catalytic Mechanism of Triose Phosphate Isomerase
Berg, Tymoczko & Stryer, 2012 Fig. 16.5

Avoiding Methyl Glyoxal by Triose Phosphate Isomerase
Berg, Tymoczko & Stryer, 2012 Chap 16 p. 460

Berg, Tymoczko & Stryer, 2012 Chap. 16 p.461
Voet, Voet & Pratt, 2013 Fig. 15.15

Stage 3 of Glycolysis
Berg, Tymoczko & Stryer, 2002 Fig. 16.X

Conversion (Oxidation) of GAP into
1,3-BPG
Glycolysis Step 6

G

= 6.3 kJ/mol
Berg, Tymoczko & Stryer, 2012 Chap.. 16 p. 461

Conversion of GAP into 1,3-BPG
Two steps involved: oxidation of aldehyde & joining of carboxylic acid with orthophosphate

G

= 6.3 kJ/mol
Glycolysis Step 6
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 461

Enzyme active site
Glycolysis Step 6
Voet, Voet &Pratt 2013 Fig. 15.9

Glyceraldehyde Oxidation Free Energy Profile
Berg, Tymoczko & Stryer, 2012 Fig. 16.6
Berg, Tymoczko & Stryer, 2012 Fig. 16.6

Glycolysis Step 7

G

= -18.5 kJ/mol
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 463

Reaction
Mechanism
Glycolysis Step7
Voet & Voet Biochemistry 2008 p. 499

IMPORTANT: This refers to the formation of ATP from a high phosphoryl transfer potential substrate.
1,3-bisphosphoglycerate (1,3-BPG) in the phosphoglycerate kinase reaction of glycolysis is such an example.

Rearrangement of 3-phosphoglycerate
Glycolysis Step 8

G

= 4.4 kJ/mol
Voet, Voet, & Pratt, 2013 Chap 15. p. 486

Phosphoglycerate Mutase Reaction Mechanism
Lehninger 2000 Fig 15.6
Voet, Voet & Pratt 2008 Fig p500

Enzyme active site
Glycolysis
Step 8 Voet & Voet Biochemistry 2013 Fig. 15.12

Dehydration of 2-phosphoglycerate
Glycolysis Step 9

G

= 7.5 kJ/mol
Voet, Voet, & Pratt 2012 Chap. 15 p. 487

Dephosphorylation of Phosphoenolpyruvate
Glycolysis Step 10

G

= -31.4 kJ/mol
Berg, Tymoczko & Stryer, 2002 Fig. 16.3;
2013 Chap 15 p. 465

Bhagavan 2001 Biochemistry Table 13.2

NADH Regeneration
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 466

Alcoholic Fermentation
Voet, Voet & Pratt 2013 Fig 15.16
Voet, Voet & Pratt 2013 Fig 15.18

Lactic Acid Fermentation
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 468

Redox Balance of NADH needed to Maintain
Glycolysis
Berg, Tymoczko & Stryer, 2012 Fig. 16.11

NAD + -Binding Domain of Dehydrogenases
Berg, Tymoczko & Stryer, 2012 Fig. 16.12

Voet, Voet , & Pratt 2013 Fig 15.26

Galactose and Fructose Entry Points in Glycolysis
Berg, Tymoczko & Stryer, 2012 Fig. 16.13

Voet, Voet & Pratt 2013 Fig 15.27

Voet, Voet & Pratt 2013 Fig 15.28

Feeder Pathways: Entry of Glycogen, Starch, Disaccharides and hexoses into preparatory stage of Glycolysis
Lehninger 2000 Fig 15.11

The metabolic flux through the glycolytic pathway must be adjusted to respond to internal and extracellular conditions.
IMPORTANT - Two major cellular needs regulate the rate of glucose conversion into pyruvate:
1) The production of ATP.
2) The production of building blocks for synthetic reactions.
In metabolic pathways, enzymes catalyzing essentially irreversible reactions are potential sites for control.
•These enzymes are regulated by allosteric effectors that reversibly bind to the enzyme or by covalent modification (meaning? E.g. phosphorylation).
•These enzymes are also subject to regulation by transcription in response to metabolic loads (demands).

Regulation of
Flux Through a
Multistep
Pathway
Lehninger 2000 Fig 15.16

Cumulative standard and actual free energy changes for the reactions of glycolysis
Horton et al 2012 Fig 11.12
Voet , Voet, & Pratt 2013 Table 15.1

For mammals, phosphofructokinase is the most important control element in the glycolytic pathway.
Berg, Tymoczko, & Stryer 2012 Fig 16.16
Voet, Voet & Pratt 2013 Fig 15.23

Effect of F-2,6-BP and ATP
Berg, Tymoczko & Stryer, 2012 Fig. 16.20

Berg, Tymoczko & Stryer, 2012 Fig. 16.32

Glycogen
Phosphorylase of
Liver as a Glucose
Sensor
Lehninger 2000 Fig 15.19

Phosphofructokinase Control
Summary of Regulatory Factors Affecting PFK
Lehninger 2000 Fig 15.18

Hexokinase is inhibited by Glucose –6-P (its product).
Indicates that the cell has sufficient energy supply. This will leave glucose in the blood.
Special case of liver: glucokinase (an isozyme) not inhibited by glucose-6-P. Has a 50-fold LOWER affinity for glucose.
Functions to provide glucose-6-P for glycogen synthesis.
Lower affinity means that hexokinase (muscle, brain) has first call on available glucose.

Pyruvate kinase controls the outflow from the glycolysis pathway. It is the third irreversible step. This final step yields ATP and pyruvate.
Several mammalian isozymes of tetramer enzyme:
L-form predominates in liver
M-form predominates in muscle and brain
Berg, Tymoczko & Stryer, 2012 Fig. 16.21