Chapter 19
Glycolysis
based on
Biochemistry, 4/e
by
Reginald Garrett and Charles Grisham
All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company,
6277 Sea Harbor Drive, Orlando, Florida 32887-6777
Outline
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
GLYCOLYSIS
Stepwise degradation of glucose
Occurs in the cytosol
Basically anaerobic process
Principal steps occur with no
requirement for oxygen
First two billion years of
biological evolution on earth
Overview of Glycolysis
(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
COUPLED REACTIONS
(in anaerobic route of glycolysis)
Phosphorylation
To synthesize ATP using the metabolic free energy
contained in the glucose molecule would be to convert
glucose into one (or more) of the high-energy
phosphates that have standard-state free energies of
hydrolysis more negative than that of ATP.
synthesized easily from glucose
Phosphoenolpyruvate
1,3-bisphosphoglycerate
acetyl phosphate.
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
Hexokinase
1st step in glycolysis
ΔG large, negative (-33kJ/mol) under cellular conditions
Hexokinase (and glucokinase) act to
phosphorylate glucose and keep it in
the cell
Km for glucose is 0.1 mM; efficient at
cell concentration of 4 mM glucose
So hexokinase is normally active!
Hexokinase is regulated - allosterically
inhibited by (product) glucose-6-P - but
is not the most important site of
regulation of glycolysis - Why?
Glucokinase (in the liver)
1st step in glycolysis
ΔG large, negative (-33kJ/mol) under cellular conditions
Glucokinase (Kmglucose = 10 mM) only turns on when
the cell is rich in glucose
Highly specific for glucose
Hexokinase coverts to glucokinase at high glucose
concentration.
Inducible enzyme – the amount present is controlled
by insulin
Glucose-6-phosphate common to several
metabolic pathways; hence, is a branch point in
glucose metabolism
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
Rx 3: Phosphofructokinase (PFK)
PFK is the committed step
in glycolysis!
The second priming
reaction of glycolysis
Committed step and
large, neg ΔG (-18.8
kJ/mol in erythrocytes)
means PFK is highly
regulated
Most important site of
regulation
Rx 3: Phosphofructokinase (PFK)
PFK increases activity when energy status is low
PFK decreases activity when energy status is high
Phosphofructokinase with ADP shown in white and
fructose-6-P in red.
ATP inhibits, AMP reverses inhibition
Citrate is also an allosteric inhibitor
Fructose-2,6-bisphosphate is allosteric activator
FIGURE 19.8 ● At high [ATP],
phosphofructokinase (PFK)
behaves cooperatively, and the
plot of enzyme activity versus
[fructose-6-phosphate] is
sigmoid.
High [ATP] thus inhibits PFK,
decreasing the enzyme’s
affinity for fructose- 6phosphate.
FIGURE 19.9 ●
Fructose-2,6bisphosphate activates
phosphofructokinase,
increasing the affinity
of the enzyme for
fructose-6-phosphate
and restoring the
hyperbolic
dependence of
enzyme activity on
substrate.
FIGURE 19.10 ●
Fructose-2,6bisphosphate
decreases the
inhibition of
phosphofructokinase
due to ATP.
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
Rx 5: Triose Phosphate Isomerase
DHAP converted to Gly-3-P
An ene-diol mechanism
Active site Glu acts as general base
Triose phosphate isomerase is a near-perfect enzyme
Catalytic perfection – turnover number near the diffusion limit
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
1,3
- BPG
Phosphoenolpyruvate
RxGly-3P
6: Gly-3-Dehydrogenase
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 involves covalent catalysis and a nicotinamide
coenzyme
Formation of a covalent intermediate in the glyceraldehyde-3phosphate dehydrogenase reaction. Nucleophilic attack by a
cysteine OSH group forms a covalent acylcysteine intermediate.
Following hydride transfer to NAD, nucleophilic attack by
phosphate yields the product, 1,3-bisphosphoglycerate.
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
FIGURE 19.21 ● Formation and decomposition of 2,3-bisphosphoglycerate.
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 2,3-BPG is required to phosphorylate
His
Rx 9: Enolase
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
Rx 10: Pyruvate Kinase
PEP to Pyruvate makes ATP
These two ATP (from one glucose) can be viewed as the "payoff"
of glycolysis
Large, negative ΔG - regulation!
Allosterically activated by AMP, F-1,6-bisP
Allosterically inhibited by ATP and acetyl-CoA
Understand the keto-enol equilibrium of Pyruvate
FIGURE 19.28 ● The
conversion of
phosphoenolpyruvate
(PEP) to pyruvate may be
viewed as involving two
steps: phosphoryl transfer
followed by an enol-keto
tautomerization. The
tautomerization is
spontaneous (ΔG° = 35–
40 kJ/mol) and accounts
for much of the free
energy change for PEP
hydrolysis.
The Fate of NADH and Pyruvate
Aerobic or anaerobic??
Pyruvate has two possible fates:
aerobic: citric acid cycle
anaerobic: LDH makes lactate
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 re-oxidized by lactate
dehydrogenase (LDH), providing additional NAD+ for more glycolysis
Energetics of Glycolysis
The elegant evidence of
regulation!
Standard state ∆G values
are scattered: + and • ∆G in cells is revealing:
Most
values near zero
3 of 10 Rxns have large,
negative ∆ G
negative ∆G rxns are
sites of regulation!
Large
Other Substrates for
Glycolysis
Fructose, mannose and galactose
Fructose and mannose are routed into
glycolysis by fairly conventional means.
Galactose is more interesting - the Leloir
pathway "converts" galactose to glucose sort of....
Compartmentalization
of glycolysis, the citric
acid cycle, and
oxidative
phosphorylation.
0
