Molecular Biochemistry I

Glycolysis and Fermentation

Contents of this page:
Glycolysis pathway reactions
Summary of pathway
Fermentation
Regulation of glycolysis
Glycolysis Pathway
The Glycolysis pathway is described below and summarized in Fig. 17.3 p. 584 of
Biochemistry, by Voet & Voet, 3rd Edition.
The reactions of Glycolysis take place in the cytosol of cells.
Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate. Initially,
there is energy input corresponding to cleavage of two ~P bonds of ATP.
1. Hexokinase catalyzes: glucose + ATP
The Hexokinase reaction
involves nucleophilic attack of
the C6 hydroxyl oxygen of
glucose on the phosphorous of
the terminal phosphate of ATP.
ATP binds to the enzyme as a
complex with Mg
++
.
The positively charged Mg
++
interacts with negatively charged
phosphate oxygen atoms of ATP,
providing charge compensation and
promoting a favorable conformation
of ATP at the active site. (See also
diagram p. 585.)
The reaction catalyzed by
Hexokinase is highly spontaneous.
A phosphoanhydride bond of ATP
(~P) is cleaved. The phosphate ester
formed in glucose-6-phosphate has
a lower G of hydrolysis.
Induced fit: Glucose binding to Hexokinase stabilizes
a conformation in which
The C6 hydroxyl of the bound glucose is close
to the terminal phosphate of ATP, promoting
catalysis.
Water is excluded from the active site. This
prevents the enzyme from catalyzing ATP
hydrolysis, rather than transfer of phosphate to
glucose.
It is a common motif for an enzyme active site to be
located at an interface between protein domains that are
connected by a flexible hinge region. The structural
flexibility allows access to the active site, while
permitting precise positioning of active site residues,
and in some cases exclusion of water, as substrate
binding promotes a particular conformation.
E O E 4 E EC M 4
4 E O E O 4 W E U U M4 4E
O EO 4 W 4E
The Phosphoglucose Isomerase
mechanism involves acid/base
catalysis, with ring opening,
isomerization via an enediolate
intermediate, and then ring
closure (diagram p. 587). A
similar reaction catalyzed by
Triose Phosphate Isomerase is
presented in more detail below.
E O E M4 4E 4
M4 4E O E O 4 *

This highly spontaneous
reaction has a mechanism
similar to that of
Hexokinase.
The Phosphofructokinase
reaction is the rate-
limiting step of
Glycolysis. The enzyme
is highly regulated, as
will be discussed later.
E 4
M4 4E O EO 4 U ME
4E O EO 4 M
O EO 4
The Aldolase reaction is an
aldol cleavage, the reverse of
an aldol condensation.
Note that carbon atoms are
renumbered in reaction
products.
A lysine residue at the active site of
the Aldolase enzyme functions in
catalysis.
The keto group of fructose-1,6-bisP
reacts with the -amino group of the
active site lysine, to form a
protonated Schiff base intermediate.
Cleavage of the bond between C3 and
C4 follows. (See p. 590).
*M E EO 4 EC M W* 4
W M C O
ME 4E O EO 4 WU 4E U U
M O EO 4 W E
E E4 4 MEC M
O EO 4 * O4 M 4C E
4 4 WC O EM 4 * M 4 E +EM
ME 4E O EO 4 44 M CE+ E
M O EO 4 4 O4
4 OE4 E4 M 4 E E 4 ME4 O4
4
The ketose/aldose conversion of
TIM involves acid/base catalysis,
and is thought to proceed via an
enediol intermediate, as with
Phosphoglucose Isomerase,
Active site Glu and His residues
are thought to extract and donate
protons during catalysis.
2-Phosphoglycolate is an example of a transition
state analog that binds tightly at the active site of
Triose Phosphate Isomerase. This inhibitor of
catalysis by TIM is similar in structure to the
proposed enediolate intermediate.
TIM is considered a "perfect enzyme", because the
reaction rate is limited only by the rate at which
substrate collides with the enzyme.
The structure of Triose Phosphate Isomerase is an
barrel, or TIM barrel.
In an barrel there are 8 parallel -strands
surrounded by 8 -helices. Short loops connect
alternating -strands and -helices.
TIM barrels serve as scaffolds for active site residues in a
diverse array of enzymes. Residues that form the active
site are always located at the same end of the barrel,
associated with the C-terminal ends of -strands and the
loops connecting these to -helices.
There is debate over whether the many different TIM
barrel enzymes are evolutionarily related, since in spite of
the structural similarities there is tremendous diversity in
catalytic functions of these enzymes and little sequence
homology.
Explore at right the structure of the Triosephosphate
Isomerase (TIM) homodimer, with the transition state
inhibitor 2-phosphoglycolate bound to one of the TIM
monomers.
Note the structure of the TIM barrel, and the loop that
forms a lid that closes over the active site after binding of
the substrate.
Triose Phosphate Isomerase
M O EO 4
ME 4
M O EO 4


U O EO E M 4

Exergonic oxidation of the aldehyde in

glyceraldehyde-3-phosphate, to a
carboxylic acid, drives formation of an acyl
phosphate, a "high energy" bond (~P), in
1,3-bisphosphoglycerate.
This is the only step in Glycolysis in which
NAD
+
is reduced to NADH.
A cysteine thiol at the active site of
Glyceraldehyde-3-phosphate
Dehydrogenase has a role in catalysis (p.
596).
The aldehyde of glyceraldehyde-3-
phosphate reacts with the active site
cysteine thiol to form a thiohemiacetal
intermediate. Oxidation to a carboxylic
acid (in a "high energy" thioester) occurs,
as NAD
+
is reduced to NADH.
The "high energy" acyl thioester is
attacked by P
i
to yield the acyl phosphate
(~P) product.
Recall that NAD
+
accepts 2 e
-
plus one H
+
(a hydride)
in going to its reduced form.
E O E M 4 C 4
O EO E M 4 U O E
O E M 4 *
This transfer of phosphate to ADP, from the
carboxyl group on 1,3-bisphosphoglycerate, is
reversible (low G), since one ~P bond is
cleaved and another is synthesized. The
enzyme undergoes a substrate-induced
conformational change similar to that of
Hexokinase.
E O E M 4 44 4
O EO E M 4 U O EO E
M 4
Phosphate is shifted from the hydroxyl on C3
of 3-phosphoglycerate to the hydroxyl on C2.
An active site histidine side-chain
participates in phosphate transfer, by
donating and accepting the phosphate. The
process involves a 2,3-bisphosphate
intermediate.
The Phosphoglycerate Mutase reaction is illustrated in the
animation at right.
of Phosphoglycerate
Mutase
E 4 O EO E M
4 U O EO E EO M4+ 4
This dehydration reaction
is Mg
++
-dependent.
2 Mg
++
ions interact with
oxygen atoms of the
substrate carboxyl group
at the active site. The Mg
+
+
ions help to stabilize the
enolate anion intermediate
that forms when a lysine
side-chain amino group
extracts a proton from
carbon #2.
M 4+ 4 C 4 O EO
E EO M4+ 4
This transfer of phosphate from PEP
to ADP is spontaneous. PEP has a
larger G of phosphate hydrolysis
than ATP, because removal of
phosphate from PEP yields an
unstable enol, that spontaneously
converts to the keto form of pyruvate
(p. 602). Required inorganic cations
K
+
and Mg
++
bind to anionic residues
at the active site of Pyruvate Kinase.
Summary of Glycolysis:
The pathway continues from
glyceraldehyde-3-phosphate. Recall
that there are two glyceraldehyde-3-
phosphate per glucose metabolized.

Balance sheet for high energy bonds of
ATP:
2 ATP expended
4 ATP produced (2 from each of
two 3C fragments from
glucose)
Net production of 2 ~P bonds
of ATP per glucose.
E 4 WEC 44

4 E



ME EM C O M4+ 4 OME 4
E E 4E + CM
4 OME 4 E
CM M E + 4 M
O M 4EM 4 OME 4 4 E E C4
4 E *
MC 4 4 E
ME EM C M O M 4EM
* C44 M E OME 4
E 4 ME4 EC E4 M M 4
E 4

EM 4 M
O EO 4 ME M
4 E W E+ 4 M E
O M4+ 4 E+ M4 4E CEM
M 4 ECOE4
* ECO 4 O 4 4 E
4 M E 4 E E
MC 4 4 E
For example, Lactate Dehydrogenase catalyzes
reduction of the keto group in pyruvate to a
hydroxyl, yielding lactate, as NADH is oxidized to
NAD
+
.
Lactate, in addition to being an end-product of
fermentation, serves as a mobile form of nutrient
energy, and possibly as a signal molecule in
mammalian organisms. Cell membranes contain
carrier proteins that facilitate transport of lactate.
Skeletal muscles ferment glucose to lactate
during exercise, when the exertion is brief
and intense. Lactate released to the blood
may be taken up by other tissues, or by
skeletal muscle after exercise, and converted
via Lactate Dehydrogenase back to
pyruvate, which may be oxidized in Krebs
Cycle or (in liver) converted to back to
glucose via gluconeogenesis.
Lactate serves as a fuel source for cardiac
muscle as well as brain neurons.
Astrocytes, which surround and protect
neurons in the brain, ferment glucose to
lactate and release it. Lactate taken up by
adjacent neurons is converted to pyruvate
that is oxidized via Krebs Cycle.
Some anaerobic organisms
metabolize pyruvate to ethanol,
which is excreted as a waste
product. NADH is converted to
NAD
+
in the reaction catalyzed by
Alcohol Dehydrogenase.
Thiamine pyrophosphate, the
cofactor for Alcohol
Dehydrogenase, is discussed
elsewhere.
MC 4 4 E 4 MEC 4 E 4E
4 4 WEC 44

4 E
ME 4 E C E 4 E
E O M O E E *

< 4 4 E E E

Glycolysis Enzyme *
G
o
'
(kJ/mol)
G
(kJ/mol)
Hexokinase -20.9 -27.2
Phosphoglucose Isomerase +2.2 -1.4
Phosphofructokinase -17.2 -25.9
Aldolase +22.8 -5.9
Triosephosphate Isomerase +7.9 negative
Glyceraldehyde-3-phosphate Dehydrogenase, & Phosphoglycerate
Kinase
-16.7 -1.1
Phosphoglycerate Mutase +4.7 -0.6
Enolase -3.2 -2.4
Pyruvate Kinase -23.0 -13.9
1 4 4 4 MEC 1E 4 1E 4 W
E C 4M M 4 E E E
=EM O
4 4 ME4 4 E O 4 M
4 4 E4ME E 4 C 4 4
4 OE4 E4 M 4 E
E EO E M4 4E
M4+ 4 C
CE E4ME E C 4 E C +E+
M 4 4EM 4 E + M E 4
M 4 E E O 4 4 4M 4 EM 4
MC 4 4E 4 4
E E4ME EM 4 4
E 4 E EM C E 4 +E
+ EMCE 4 + 4
C + M + C EM ME C
4 E C 4 C 4 EE
+ + M E4 E 44M 4 4
4 E * 4 E E4ME O
+E+ + M EC O 4 E E
E4ME EMCE 4 + 4
4 4 4
E E 4 E E
E 4 M4 4 O 4 E
O 4 4 4 OME 4
4 4 E O EO 4
ECO 4 4 E 4 4 4 + 4

E4 M 4 M 4 E 4 O
M 4 4 E 4 C
4M O 4 E O EO EM 4
4 OM + 4 4 E 4 E MM M
ME 4 4 4 E E E 4M
4 4 E4 E4 4 4E 4C4
4 4 E MEC 4 EE 4 E
O EO 4 # 4 4 CO
4 E + M 4 E E E
4 + M
4 E C EM 4 E
4 4 4 4 + E 4
4 E #
4 E 4 EMCE OME
4 M OE 4E EE 4 E
4 + 4 E + M E 4M M
O4 E E 4 4 4 E 4
4 E C
4 E E4 4 4 4E OME 4
4 4 E 4 E O EO 4
* + M 4 4O O EO EM
4 4 E + + M 4 E
O EO 4 #
C + M 4 E 4 4 4E
4 E 4 E M 4 4EM
OME4 W C< * M 4 E E 4 E
4E C< + M
M 4 C 4 E 4 4 OME+ C
C EM CE 4 4 M 4 E 4 E
O EO EM 4 E
Glucokinase, with its high KM for
glucose, allows the liver to store
glucose as glycogen in the fed state
when blood [glucose] is high.
The liver enzyme Glucose-6-
phosphatase catalyzes hydrolytic
release of P
i
from glucose-6-
phosphate. Thus glucose is released
from the liver to the blood as
needed to maintain blood [glucose].
The enzymes Glucokinase and
Glucose-6-phosphatase, both found
in liver but not in most other body
cells, allow the liver to control
blood [glucose].
M4+ 4 C 4 4 4 O E 4
E O 4 E4ME + M
O M4 CE 4 4 E E 4 CE44 E
C
4 E # 4 + M
4 4M M O4 E 4EM M E M
4 M OE + C 4 OME
4 W < 4E 4M MM 4E
4 4 4 M 4 4 + 4 4M
M O4 E E 4 EM M4+ 4 C

* 4 4 E+ M4 EC E 4
4 E 4E O M4+ 4
C 4 E 4E 4
E 4 C OM 4MEM EM 4
E 44
EM E 4 MC M 4EM
Phosphofructokinase is usually the rate-limiting step of the Glycolysis pathway.
Phosphofructokinase catalyzes:
fructose-6-phosphate + ATP
EO E M4 4E W C E4 M 4 *
4 E E 4M 4 E 4 4 4M 4 * E 4 4 4 + 4 4 E 4M 4 E * E 4 E M 4
4 4 4 E EMC 4 E * 4 E EMC 4 E E EO E M4 4E E M 4 EM 4 E4 M 4 4M 4 M4 4E O EO 4
CE O E M 4 E M 4 E M4 4E O E O 4 #
OM 4 4 4 +
E 4 M 4 + * ME
4 E 4 4 E * #

4 E E EO E M4 4E 4
M 4 C 4 4 O E E
* # OM + 4 M E E 4
E O 4 E C ME 4E C
* 4 CEM 4 4 4E 4 4E 4
EM 4 E E * O 4
4 W M C E 4 M E 4 O 4
E+
44 E M
1. Explore in the Biochemistry Simulations tutorials at right
concepts of enzyme kinetics and enzyme regulation relevant
to this class:
In the module on Glucokinase, compare the
dependence on [glucose] of catalysis by Glucokinase
and Hexokinase.
In the module on Phosphofructokinase,
explore effects of varied [ATP] at zero [fructose-2,6-
bisphosphate]. (The activator fructose-2,6-
bisphosphate will be discussed in the section on
gluconeogenesis.)
Note: Hold down the
Control key while
clicking on the above
icon.
4 OEM 4 EE C 4 M
+ EO E M
=
E 4 M 4 + 444EM
E 4 E 4 44M E M 4 E
4 E O 4
4 O 4 O
E 4 C 4 E E
4 O E 4 O 4
EO M 4 ' E M
4 M M+
Additional
material
on
Glycolysis:
Readings,
Test
Questions,
& Tutorial