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