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GLYCOLYSISStudent Edition 5/30/13 version
Pharm. 304 Biochemistry
Fall 2014
Dr. Brad Chazotte 213 Maddox Hall
chazotte@campbell.edu
Web Site:
http://www.campbell.edu/faculty/chazotte
Original material only ©2000-14 B. Chazotte
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
An Energy Conversion Pathway Used by Many Organisms
Glycolysis:
• 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
Glucose + 2NAD+ + 2ADP + 2Pi
2 pyruvate + 2 NADH + 2H+ + 2ATP + 2H2O
Conversion of glucose into pyruvate: G1 = -146 kJ mol-1
Glucose + 2NAD+ 2 pyruvate + 2 NADH + 2H+
Formation of ATP from ADP and Pi G2 = 2 (30.5)= 61 kJ mol-1
2ADP + 2Pi 2ATP + 2H2O
Gs = G1 + G1 = -146 kJ mol-1 + 61 kJ mol-1 = -85 kJ mol-1
Overall Reaction of Glycolysis
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
Voet, Voet & Pratt 2013 Fig 15.16
O O -
CH3 C C O
Pyruvate
Catabolism via PyruvateMajor Glucose Utilization Pathways in Cells of Higher Plants and Animals
Lehninger 2000 Fig 15.1
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-
FERMENTATION
Berg, Tymoczko & Stryer, 2012 Table. 16.1
Reactions of Glycolysis
Berg, Tymoczko & Stryer, 2002 Fig. 16.3
1. Trap and destabilize
2. Cleave 6-C into two 3-C molecules
3. Generate ATP
Schematic of the
Glycolysis Pathway
Hexose stage
Triose stage
Horton 2-stage
Berg, Tymoczko & Stryer, 2002 Fig. 16.X
Stage 1 of Glycolysis Detail
Horton, 2002 Fig 11.3Glycolysis Step 1 G= -16.7 kJ/mol
Conversion of Glucose by Hexokinase
carbon numbering
mechanism
Lehninger 2000 Fig 15.1
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: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.
Berg, Tymoczko & Stryer, 2012 Fig. 16.3
Hexokinase Structure &
Glucose Binding
Voet, Voet & Pratt , 2008 Fig. 15.2
Yeast HexokinaseTwo 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 Chap 16 p. 457Glycolysis Step 2
G=1.7 kJ/mol
Isomerization of Glucose-6-P to Fructose-6-P
Phosphoglucose Isomerase Mechanism
Voet, Voet & Pratt 20012 Fig. 15.3
Enzyme active site
Glycolysis Step 2
Glu?
Lys?
Berg, Tymoczko & Stryer, 2012 Chap 16Glycolysis Step 3
G= -14.2 kJ/mol
Phosphorylation of Fructose 6-P
Berg, Tymoczko & Stryer, 2002 chap 16.
Stage 2 of Glycolysis
Berg, Tymoczko & Stryer, 2002 Chap. 16
Berg, Tymoczko & Stryer, 2012 chap 16 p. 458Glycolysis Step 4
G=23.8 kJ/mol
Cleavage of Fructose 1,6-biphosphate by Aldolase
Aldolase Reaction: Glycolysis Rx #4
Glycolysis Step 4Voet, Voet & Pratt 2013 15 p. 478
Base-catalyzed Aldol Cleavage Mechanism
Voet, Voet & Pratt 2013 Fig. 15.4Glycolysis
Aldolase Mechanism
Voet, Voet & Pratt 2013 Fig. 15.5
The cleavage by aldolase of F1,6BP stabilizes the enolate intermediate via increased electron delocalization.
Berg, Tymoczko & Stryer, 2002 Chap 16.
Stage 2 of Glycolysis
End of “stage I ” in Voet, Voet & Pratt
Berg, Tymoczko & Stryer, 2002 Fig. 16.3Glycolysis Step 5
G=7.5kJ/mol
Isomerization of Dihdroxyacetone phosphate
Lehninger 2000 Fig 15.4
Isomerization of DHAP with Carbon #s
Triose Phosphate Enzyme Mechanism
Cunningham 1978, p343
Triose Phosphate Isomerase Rx Proposed Mechanism
Voet & Voet Biochemistry 1995 Fig.16.10
Glycolysis Step 5
Berg, Tymoczko & Stryer, 2012 Fig. 16.5
Catalytic Mechanism of Triose Phosphate Isomerase
Berg, Tymoczko & Stryer, 2012 Chap 16 p. 460
Avoiding Methyl Glyoxal by Triose Phosphate Isomerase
Berg, Tymoczko & Stryer, 2012 Chap. 16 p.461
Stage 3 Glycolysis Overview
Voet, Voet & Pratt, 2013 Fig. 15.15
Berg, Tymoczko & Stryer, 2002 Fig. 16.X
Stage 3 of Glycolysis
Berg, Tymoczko & Stryer, 2012 Chap.. 16 p. 461Glycolysis Step 6
G= 6.3 kJ/mol
Conversion (Oxidation) of GAP into 1,3-BPG
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 461Glycolysis Step 6
Two steps involved: oxidation of aldehyde & joining of carboxylic acid with orthophosphate
G= 6.3 kJ/mol
Conversion of GAP into 1,3-BPG
Glyceraldehyde-3-phosphate Dehydrogenase Mechanism
Voet, Voet &Pratt 2013 Fig. 15.9
Enzyme active site
Glycolysis Step 6
Berg, Tymoczko & Stryer, 2012 Fig. 16.6
Glyceraldehyde Oxidation Free Energy Profile
Berg, Tymoczko & Stryer, 2012 Fig. 16.6
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 463Glycolysis Step 7
G= -18.5 kJ/mol
Phosphoglycerate Kinase
Phosphoglycerate Kinase Reaction
Voet & Voet Biochemistry 2008 p. 499
Glycolysis Step7
MechanismReaction
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.
Voet, Voet, & Pratt, 2013 Chap 15. p. 486Glycolysis Step 8
G= 4.4 kJ/mol
Rearrangement of 3-phosphoglycerate
Lehninger 2000 Fig 15.6
Phosphoglycerate Mutase Reaction Mechanism
Voet, Voet & Pratt 2008 Fig p500
Phosphoglycerate Mutase Proposed Mechanism
Voet & Voet Biochemistry 2013 Fig. 15.12
Enzyme active site
Glycolysis Step 8
Voet, Voet, & Pratt 2012 Chap. 15 p. 487Glycolysis Step 9
G= 7.5 kJ/mol
Dehydration of 2-phosphoglycerate
Glycolysis Step 10 G= -31.4 kJ/mol
Dephosphorylation of Phosphoenolpyruvate
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
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 466
The Redox Balance in Glycolysis
NADH Regeneration
Alcoholic Fermentation
Voet, Voet & Pratt 2013 Fig 15.18Voet, Voet & Pratt 2013 Fig 15.16
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 468
Lactic Acid Fermentation
Berg, Tymoczko & Stryer, 2012 Fig. 16.11
Redox Balance of NADH needed to Maintain Glycolysis
Berg, Tymoczko & Stryer, 2012 Fig. 16.12
NAD+-Binding Domain of Dehydrogenases
Entry of other Hexoses into Glycolysis
Voet, Voet , & Pratt 2013 Fig 15.26
Berg, Tymoczko & Stryer, 2012 Fig. 16.13
Galactose and Fructose Entry Points in Glycolysis
Fructose Metabolism
Voet, Voet & Pratt 2013 Fig 15.27
Galactose Metabolism
Voet, Voet & Pratt 2013 Fig 15.28
Lehninger 2000 Fig 15.11
Feeder Pathways: Entry of Glycogen, Starch, Disaccharides and hexoses into preparatory stage of Glycolysis
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).
Lehninger 2000 Fig 15.16
Regulation of Flux Through a
Multistep Pathway
Cumulative standard and actual free energy changes for the reactions of glycolysis
Horton et al 2012 Fig 11.12Voet , Voet, & Pratt 2013 Table 15.1
Phosphofructokinase Control
For mammals, phosphofructokinase is the most important control element in the glycolytic pathway.
Berg, Tymoczko, & Stryer 2012 Fig 16.16Voet, Voet & Pratt 2013 Fig 15.23
Berg, Tymoczko & Stryer, 2012 Fig. 16.20
Phosphofructokinase Control IIEffect of F-2,6-BP and ATP
Berg, Tymoczko & Stryer, 2012 Fig. 16.32
Glucagon Signal Pathway
Lehninger 2000 Fig 15.19
Glycogen Phosphorylase of
Liver as a Glucose Sensor
Lehninger 2000 Fig 15.18
Phosphofructokinase Control Summary of Regulatory Factors Affecting PFK
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.
Berg, Tymoczko & Stryer, 2012 Fig. 16.21
Pyruvate Kinase Control
Several mammalian isozymes of tetramer enzyme:
L-form predominates in liver
M-form predominates in muscle and brain
Pyruvate kinase controls the outflow from the glycolysis pathway. It is the third irreversible step. This final step yields ATP and pyruvate.
End of Lectures