232
chapter 13
Carbohydrate Metabolism I: Glycolysis and TCA Cycle
co c r
I
+ E— 0P 032”
CHOPO
3
2“
+ E — OH
c h
2
o p o 32 -
2 ,3 -B ip h o s-
p h o g ly c e ra te
E—0 P 032“ + HCOPO
3
2-
I
CHjOH
2 -P h o s p h o -
g ly c e ra te
D eh yd ra tio n o f 2 -P h o sp h o g lycera te
to P h o sp h o en o lp yru va te
In this reversible reaction, dehydration of the substrate
causes a redistribution of energy to form the high-energy
compound phosphoenolpyruvate.
Mg2+
2-Phosphoglycerate -—^ phosphoenolpyruvate + H
2
O
Enolase (2-phospho-D-glycerate hydrolyase) is a homod-
imer (M.W. 88,000) that is inhibited by fluoride, with for-
mation of the magnesium fluorophosphate complex at the
active site. This property of fluoride is used to inhibit gly-
colysis in blood specimens obtained for measurement of
glucose concentration. In the absence of fluoride (or any
other antiglycolytic agent), the blood glucose concentra-
tion decreases at about 10 mg/dL (0.56 mM/L) per hour
at 25°C. The rate of decrease is more rapid in blood from
newborn infants owing to the increased metabolic activity
of the erythrocytes and in leukemia patients because of the
larger numbers of leukocytes.
Neuron-specific and non-neuron-specific enolase isoen-
zymes have been used as markers to distinguish neurons
from nonneuronal cells (e.g., glial cells that are physi-
cally and metabolically supportive cells of neurons) by im-
munocytochemical techniques. Neuron-specific enolase is
extremely stable and resistant to a number of
in vitro
treat-
ments (e.g., high temperature, urea, chloride) that inacti-
vate other enolases. The functional significance of these
isoenzymes is not known.
P h o sp h o ryla tio n o f A D P
fr o m P h o sp h o en o lp yru va te
In this physiologically irreversible (nonequilibrium) re-
action, the high-energy group of phosphoenolpyruvate is
transferred to ADP by pyruvate kinase, thereby generating
ATP (i.e., two molecules of ATP per molecule of glucose).
This reaction is the second substrate-level phosphorylation
reaction of glycolysis.
Phosphoenolpyruvate3- + ADP3- + H+ ->
pyruvate- + ATP
4
The pyruvate kinase reaction has a large equilibrium con-
stant because the initial product of pyruvate, the enol form,
rearranges nonenzymatically to the favored keto form:
C O O “
C O O “
n
I
CH
2
CH
3
E n o l-p y ru v a te
K e to -p y ru v a te
Several isoenzyme forms of pyruvate kinase are known
(M.W. 190,000-250,000, depending on the source). Each
is a homotetramer exhibiting catalytic properties consis-
tent with the function of the tissue in which it occurs.
Enzyme activity is dependent on K+ (which increases the
affinity for phosphoenolpyruvate) and Mg2+.
Pyruvate kinase is an allosteric enzyme regulated by
several modifiers. The liver isoenzyme (L-type) shows
sigmoidal kinetics with phosphoenolpyruvate. Fructose-
1
,
6
-bisphosphate is a positive modulator and decreases
Km
for phosphoenolpyruvate; ATP and alanine are neg-
ative modulators and increase
Km
for phosphoenolpyru-
vate. The former is an example of positive feed-forward
regulation, and the latter are examples of negative feed-
back regulation. Alanine, a gluconeogenic precursor, is
obtained by proteolysis or from pyruvate by amino trans-
fer (Chapter 17). The modulation of pyruvate kinase is
consistent with the function of the liver; when glucose
abounds, its oxidation is promoted, and when glucose
is deficient, its formation is favored by gluconeogenesis
(Chapter 15).
The L-type is also regulated by diet and hormones.
Fasting
or
starvation
decreases
activity,
whereas
a
carbohydrate-rich diet increases it. Insulin increases ac-
tivity, whereas glucagon decreases it. These hormones
also have reciprocal effects on gluconeogenesis, which in-
sulin inhibits and glucagon promotes. Glucagon action is
dependent on the cAMP-mediated cascade process of re-
versible phosphorylation and dephosphorylation of the en-
zyme (Chapter 30). The cAMP cascade begins with stim-
ulation of membrane-bound adenylate cycles by glucagon
to form cAMP and is followed by activation of cAMP-
dependent protein kinase, which phosphorylates pyruvate
kinase. The phospho enzyme is less active than the de-
phospho form, has a higher
Km
for phosphoenolpyruvate,
co c r
1
CHOH
I
CH
2
0 P 032“
3 -P h o s p h o g ly c e ra te
