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Biochemistry 2/e - Garrett & Grisham
Chapter 19
Glycolysis
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
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Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Outline
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19.1 Overview of Glycolysis
19.2 Coupled Reactions in Glycolysis
19.3 First Phase of Glycolysis
19.4 Second Phase of Glycolysis
19.5 Metabolic Fates of NADH and Pyruvate
19.6 Anaerobic Pathways for Pyruvate
19.7 Energetic Elegance of Glycolysis
19.8 Other Substrates in Glycolysis
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Overview of Glycolysis
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The Embden-Meyerhof (Warburg) Pathway
Essentially all cells carry out glycolysis
Ten reactions - same in all cells - but rates differ
Two phases:
– First phase converts glucose to two G-3-P
– Second phase produces two pyruvates
Products are pyruvate, ATP and NADH
Three possible fates for pyruvate
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
First Phase of Glycolysis
The first reaction - phosphorylation of glucose
• Hexokinase or glucokinase
• This is a priming reaction - ATP is consumed
here in order to get more later
• ATP makes the phosphorylation of glucose
spontaneous
• Be SURE you can interconvert Keq and
standard state free energy change
• Be SURE you can use Eq. 3.12 to generate
far right column of Table 19.1
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Hexokinase
1st step in glycolysis; G large, negative
• Hexokinase (and glucokinase) act to
phosphorylate glucose and keep it in the cell
• Km for glucose is 0.1 mM; cell has 4 mm glucose
• So hexokinase is normally active!
• Glucokinase (Kmglucose = 10 mM) only turns on
when cell is rich in glucose
• Hexokinase is regulated - allosterically inhibited
by (product) glucose-6-P - but is not the most
important site of regulation of glycolysis - Why?
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Rx 2:
Phosphoglucoisomerase
Glucose-6-P to Fructose-6-P
• 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
• Ene-diol intermediate in this reaction
• Be able to write a mechanism!
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Rx 3: Phosphofructokinase
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PFK is the committed step in glycolysis!
The second priming reaction of glycolysis
Committed step and large, neg delta G - means
PFK is highly regulated
ATP inhibits, AMP reverses inhibition
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
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Rx 4: Aldolase
C6 cleaves to 2 C3s (DHAP, Gly-3-P)
• Animal aldolases are Class I aldolases
• Class I aldolases form covalent Schiff
base intermediate between substrate
and active site lysine
• Understand the evidence for Schiff base
intermediate (box on page 622)
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Rx 5: Triose Phosphate
Isomerase
DHAP converted to Gly-3-P
• An ene-diol mechanism (know it!)
• Active site Glu acts as general base
• Triose phosphate isomerase is a nearperfect enzyme - see Table 14.5
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Glycolysis - Second Phase
Metabolic energy produces 4 ATP
• Net ATP yield for glycolysis is two ATP
• Second phase involves two very high
energy phosphate intermediates
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– 1,3 BPG
– Phosphoenolpyruvate
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Rx 6: Gly-3-Dehydrogenase
Gly-3P 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
• Mechanism is one we saw in Chapter
16 (see Figure 16.10!)
• Mechanism involves covalent catalysis
and a nicotinamide coenzyme - know it
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Rx 7: Phosphoglycerate
Kinase
ATP synthesis from a high-energy
phosphate
• This is referred to as "substrate-level
phosphorylation"
• 2,3-BPG (for hemoglobin) is made by
circumventing the PGK reaction
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Rx 8: Phosphoglycerate
Mutase
Phosphoryl group from C-3 to C-2
• Rationale for this enzyme - repositions
the phosphate to make PEP
• Note the phospho-histidine
intermediates!
• Zelda Rose showed that a bit of 2,3BPG is required to phosphorylate His
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Rx 9: Enolase
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2-P-Gly to PEP
Overall G is 1.8 kJ/mol
How can such a reaction create a PEP?
"Energy content" of 2-PG and PEP are
similar
Enolase just rearranges to a form from
which more energy can be released in
hydrolysis
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Rx 10: Pyruvate Kinase
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PEP to Pyruvate makes ATP
These two ATP (from one glucose) can be
viewed as the "payoff" of glycolysis
Large, negative G - regulation!
Allosterically activated by AMP, F-1,6-bisP
Allosterically inhibited by ATP and acetylCoA
Understand the keto-enol equilibrium of Py
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
The Fate of NADH and
Pyruvate
Aerobic or anaerobic??
• NADH is energy - two possible fates:
– If O2 is available, NADH is re-oxidized in
the electron transport pathway, making
ATP in oxidative phosphorylation
– In anaerobic conditions, NADH is reoxidized by lactate dehydrogenase (LDH),
providing additional NAD+ for more
glycolysis
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Biochemistry 2/e - Garrett & Grisham
The Fate of NADH and Py
Aerobic or anaerobic??
• Pyruvate is also energy - two possible
fates:
– aerobic: citric acid cycle
– anaerobic: LDH makes lactate
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Energetics of Glycolysis
The elegant evidence of regulation!
• See Figure 16.31
• Standard state G values are scattered:
+ and  G in cells is revealing:
– Most values near zero
– 3 of 10 Rxns have large, negative  G
• Large negative  G Rxns are sites of
regulation!
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Other Substrates for
Glycolysis
Fructose, mannose and galactose
• Fructose and mannose are routed into
glycolysis by fairly conventional means.
See Figure 18.32
• Galactose is more interesting - the Leloir
pathway "converts" galactose to glucose
- sort of....
• See Figure 16.33
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company