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GLYCOLYSIS
Student Edition
5/30/13 version
Dr. Brad Chazotte
213 Maddox Hall
[email protected]
Web Site:
http://www.campbell.edu/faculty/chazotte
Original material only ©2000-14 B. Chazotte
Pharm. 304
Biochemistry
Fall 2014
Goals
•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
Glycolysis:
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 + 2Pi
2 pyruvate + 2 NADH + 2H+ + 2ATP + 2H2O
Conversion of glucose into pyruvate:
Glucose + 2NAD+
2 pyruvate + 2 NADH + 2H+
Formation of ATP from ADP and Pi
2ADP + 2Pi
G1 = -146 kJ mol-1
G2 = 2 (30.5)= 61 kJ mol-1
2ATP + 2H2O
Gs = G1 + G1 = -146 kJ mol-1 + 61 kJ mol-1 = -85 kJ mol-1
The Glycolysis Pathway
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 OCH3
Lehninger 2000 Fig 15.1
Voet, Voet & Pratt 2013 Fig 15.16
C
C
O
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:
Fermentations produce less energy than complete
combustion with oxygen
Advantage:
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 CH3-CH2OH
Lactic Acid Fermentation: e.g. the conversion of pyruvate from
glycolysis to lactic acid in skeletal muscle. CH3-CHOH-COO-
Reactions of Glycolysis
Berg, Tymoczko & Stryer, 2012 Table. 16.1
Schematic
of the
Glycolysis
Pathway
Horton 2-stage
Hexose
stage
1. Trap and
destabilize
2. Cleave 6-C into
two 3-C molecules
3. Generate ATP
Triose
stage
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
Reaction Purposes:
mechanism
Glycolysis Step 1
G= -16.7 kJ/mol
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.
Lehninger 2000 Fig 15.1
Horton, 2002 Fig 11.3
Hexokinase
Structure &
Glucose Binding
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
Voet, Voet & Pratt , 2008 Fig. 15.2
Berg, Tymoczko & Stryer, 2012 Fig. 16.3
Isomerization of Glucose-6-P to Fructose-6-P
G=1.7 kJ/mol
Glycolysis Step 2
Berg, Tymoczko & Stryer, 2012 Chap 16 p. 457
Phosphoglucose Isomerase
Mechanism
Enzyme
active site
Lys?
Glu?
Glycolysis Step 2
Voet, Voet & Pratt 20012 Fig. 15.3
Phosphorylation of Fructose 6-P
G= -14.2 kJ/mol
Glycolysis Step 3
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
G=23.8 kJ/mol
Glycolysis Step 4
Berg, Tymoczko & Stryer, 2012 chap 16 p. 458
Aldolase Reaction: Glycolysis Rx #4
Glycolysis Step 4
Voet, Voet & Pratt 2013 15 p. 478
Base-catalyzed Aldol Cleavage
Mechanism
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
G=7.5kJ/mol
Glycolysis Step 5
Berg, Tymoczko & Stryer, 2002 Fig. 16.3
Isomerization of DHAP with Carbon #s
Lehninger 2000 Fig 15.4
Triose Phosphate Enzyme
Mechanism
Cunningham 1978, p343
Triose Phosphate Isomerase Rx Proposed
Mechanism
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
Stage 3
Glycolysis
Overview
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
G= 6.3 kJ/mol
Glycolysis Step 6
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
Glyceraldehyde-3-phosphate
Dehydrogenase Mechanism
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
Phosphoglycerate Kinase
G= -18.5 kJ/mol
Glycolysis Step 7
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 463
Phosphoglycerate Kinase Reaction
Mechanism
Reaction
Glycolysis Step7
Voet & Voet Biochemistry 2008 p. 499
SUBSTRATE-LEVEL
PHOSPHORYLATION
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
G= 4.4 kJ/mol
Glycolysis Step 8
Voet, Voet, & Pratt, 2013 Chap 15. p. 486
Phosphoglycerate Mutase Reaction Mechanism
Voet, Voet & Pratt 2008 Fig p500
Lehninger 2000 Fig 15.6
Phosphoglycerate Mutase Proposed
Mechanism
Enzyme
active site
Glycolysis
Step 8
Voet & Voet Biochemistry 2013 Fig. 15.12
Dehydration of 2-phosphoglycerate
G= 7.5 kJ/mol
Glycolysis Step 9
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
Enzymes of Glycolysis Table
Bhagavan 2001 Biochemistry Table 13.2
Channeling of Intermediates in
Glycolysis
The Redox
Balance in
Glycolysis
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
Entry of other Hexoses into
Glycolysis
Voet, Voet , & Pratt 2013 Fig 15.26
Galactose and Fructose Entry Points in Glycolysis
Berg, Tymoczko & Stryer, 2012 Fig. 16.13
Fructose Metabolism
Voet, Voet & Pratt 2013 Fig 15.27
Galactose Metabolism
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
Control of the Glycolytic
Pathway
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
Phosphofructokinase Control
For mammals, phosphofructokinase is the most important control element in the glycolytic
pathway.
Voet, Voet & Pratt 2013 Fig 15.23
Berg, Tymoczko, & Stryer 2012 Fig 16.16
Phosphofructokinase Control II
Effect of F-2,6-BP and ATP
Berg, Tymoczko & Stryer, 2012 Fig. 16.20
Glucagon Signal Pathway
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 Control
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 Control
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
End of Lectures