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chapter 13
Carbohydrate Metabolism I: Glycolysis and TCA Cycle
N icotinam ide
A denine
F I G U R E 1 3 -3
Nicotinamide adenine dinucleotide (NAD+).
two molecules of D-glyceraldehyde 3-phosphate from one
molecule of fructose-1,
6
-bisphosphate. This reaction con-
cludes the first phase of glycolysis, during which one
molecule of glucose yields two triose phosphates and two
ATP molecules are consumed. The second phase of gly-
colysis consists of energy conservation and begins with
the oxidation of glyceraldehyde 3-phosphate.
D eh yd ro g en a tio n o f G lycera ld eh yd e 3 -P h o sp h a te
In a reversible reaction, glyceraldehyde 3-phosphate is
converted by glyceraldehyde phosphate dehydrogenase to
an energy-rich intermediate, 1,3-bisglycerophosphate (or
3-phosphoglyceroyl-phosphate).
D-Glyceraldehyde 3-phosphate2” + NAD+ + P2” ^
1,3-bisphosphoglycerate4“ + NADH + H+
The rabbit skeletal enzyme is a homotetramer (M.W.
146,000). Each subunit contains a binding region for
glyceraldehyde 3-phosphate and another for nicotinamide
adenine dinucleotide (NAD+). NAD+, a cosubstrate of
this reaction, participates in many hydrogen transfer re-
actions. NAD+ (Figure 13-3) contains the vitamin nicoti-
namide (Chapter 38). Nicotinamide can also be obtained
from the amino acid tryptophan. An -SH group present at
the active site plays a prominent role in the catalysis, and
iodoacetate (ICH
2
COO” ) and heavy metals (e.g., Hg2+,
Pb2+), which react with sulfhydryl groups covalently, in-
activate the enzyme (E):
E-SH + ICH2COO
-> E-S-CH
2
COO” + HI
The reaction mechanism of glyceraldehyde-3-phosphate
dehydrogenase has several steps (Figure 13-4). First, the
enzyme-SH group attacks the carbonyl group of the sub-
strate to form a thiohemiacetal, which is then oxidized
to a thioester by transfer of a hydride ion (a hydrogen
with two electrons, H” ) to an enzyme-bound NAD+, with
concurrent release of a proton (H+). Thus, in effect, two
hydrogen atoms are removed from the substrate and the
overall NAD+-dependent reaction can be written as
Reduced substrate + NAD+ ^
oxidized substrate + NADH + H+
Once NADH is formed, its affinity for the enzyme
decreases, so that a free NAD+ displaces this NADH.
The thioester is an energy-rich intermediate, and by
phosphorolysis the high-energy 1,3-bisphosphoglycerate
is generated
with the release of the
free enzyme.
Thus, the substrate aldehyde group is oxidized to a
carboxylic acid group, with conservation of most of
the energy of oxidation in formation of the anhy-
dride bond between carboxylic and phosphoric acids. If
arsenate is used in place of phosphate, the intermedi-
ate formed is l-arseno-3-phosphoglycerate, which spon-
taneously hydrolyzes to arsenate and 3-phosphoglycerate
with release of heat. Thus, arsenate uncouples this phos-
phorylation coupled to substrate oxidation. This reac-
tion is a model for similar reactions in which energy
released by oxidation of a substrate is conserved in the
terminal phosphoanhydride bond of ATP via the forma-
tion of a high-energy intermediate (the next reaction of
glycolysis).
