208
Substrate Level Phosphorylation
The TCA cycle substrate level phosphorylation may be
examined similarly. In order for one turn of the cycle to
generate 2 CO2 from acetyl CoA, three additional oxygen
atoms must be introduced. Two are introduced from
water by citrate synthase (hydrolysis) and by fumarase
(hydration), respectively. The third is introduced from Pi
by the reversible ligase, succinyl CoA synthetase, which by
definition neither consumes nor generates water. The
water involvement in the aconitase reaction can be
ignored as it produces no net uptake or formation of
water. These newly-introduced oxygens will be lost subsequently as CO2 on later turns of the cycle.
The TCA reactions which account for the carbon and
oxygen balance may be summarized:
mediate forms such as metaphosphate anions, or by the
use of partial redox reactions. Although the overall stoichiometry of water formation might seem to suggest that
the oxygens lost from Pi in the substrate phosphorylation
steps appear in water this is not the case--ultimately these
oxygens appear in CO2. This type of 'chemical compartmentation' of water permits ATP synthesis in a membrane-free system by substrate level phosphoration.
By contrast, oxidative phosphorylation and photophosphorylation do incorporate Pi oxygen into water. In
accord with the Mitchell chemi-osmotic hypothesis these
systems require membrane-bound, anisotropic protontranslocating ATPases running in reverse to effect pyrophosphate bond synthesis.
acetyl CoA + 2 H 2 0 q- Pi + G D P ~ 2 CO2 + CoA + GTP
References
1 Horiike, K, Miura, R, Ishida, T and Nozaki, M (1996) Biochemical
Education 24, 17-20
2 Hinkle, P C, Kumar, M A, Resetar, A and Harris, D L (1991) Biochemistry 30, 3576-3582
3 Lee, C P, Gu, Q, Mitchell, R A and Ernster, L (1996) FASEB J 10,
345-350
4 Harden, A and Young, W J (1906) Proc Chem Soc 22, 283
5 Itada, N and Cohn, M (1963)J Biol Chem 238, 4026-4031
6 Alberty, R A (1994) Biochim BiophysActa 1207, 1-11
For the redox balance per acetyl CoA turned by the
cycle:
3 NADoxia+ FADoxid--O3 NmDre d -4-FADreo
Overall Reactions
Summarizing the overall reactions for the complete biologic oxidation of glucose:
PIh S0307-4412 (96)00122-7
Glycolysis
glucose+2 A D P + 2 P i ~ 2 lactate+2 H 2 0 + 2 ATP (1)
(This reaction could represent glycolysis in exercised
muscle with release of lactate to blood.)
2 lactate + 2 NADoxi~-o2 pyruvate + 2 NAI)re d
(2)
(This reaction could represent the oxidation of blood lactate by the liver or heart.)
Pyruvate dehydrognase plus TCA cycle
2 pyruvate + 2NADo,ad~ 2 acetyl CoA + 2NADre d + 2 CO2
(3)
2acetylCoA + 4H20 + 2Pi + 2GDP + 6NADox~a+ 2FADoxid
4 CO
2 -1- 2 CoA
+ 2 GTP + 6 NADre d + 2 FADre d
(4)
Oxidative phosphorylation
10 N A D r e a + 2 FADred + 3 4 A D P + 3 4 P i + 6 02
10 NADoxid + 2 FADoxid + 34 ATP + 46 H20
(5)
Sum of all reactions for combustion of glucose
glucose + 36 ADP + 2 GDP + 38 Pi + 6 02
~ 6 CO2 + 36 ATP + 2 GTP + 44 H20
Note that the 44 H20 formed is derived as follows: 34
from ATP synthase plus 12 from cytochrome oxidase plus
2 from enolase minus 4 consumed by the TCA cycle. In
summary, the stoichiometry can be deduced from the individual reactions without consideration of possible inter-
BIOCHEMICAL EDUCATION 24(4) 1996
How Many Water Molecules Produce during the
Complete Oxidation of Glucose?
Reply to Robert A Mitchell
KIHACHIRO HORIIKE,* TETSUO ISHIDA* and
RETSU MIURAt
*Department of Biochemistry
Shiga University of Medical Science
Seta, Ohtsu
Shiga 520-21
and
tDepartment of Biochemistry
Kumamoto University School of Medicine
Honjo, Kumamoto, Kumamoto 860
Japan
Mitchell's argument 1 is based on his misunderstanding of
our interpretation of the stoichiometry of water molecules
in the glucose oxidation. 2 We wish to settle the point on
the following grounds.
Stoichiometry is a 'state function' so-to-speak, so that it
is independent of the pathway, ie, the mechanisms, and
the mechanisms per se do not determine the stoichiomerry. However, the interpretation of the stoichiometry
requires mechanistic considerations. As to the metaphosphate moiety of Pi, we do not refer to the role of the
metaphosphate let alone 'unwarranted assumption
regarding a role of metaphosphate',l but we simply dissect
Pi into two separate moieties.
Mitchell's argument with respect to the P : O ratio is
irrelevant since our theory is independent of the P : O
209
ratio. In other words, it holds true whether the P : O ratio
is 3 or 2 or any other number. Different P : O ratios will
only change the number of H20 produced in oxidative
phosphorylation based on the equation, ADP +
P i ~ A T P + H 2 0 , but this number does not change the
numbers of H z O in other reactions which are important in
our considerations.
In subdividing oxidative degradation of glucose into
glycolysis, the citric acid cycle and oxidative phosphorylation, glycolysis needs to be treated up to the formation of
pyruvate rather than of lactate as stated by Mitchell, since
the latter is formed only in an anaerobic process whereas
we are dealing with aerobic degradation. In this context,
Mitchell's notion that our equation 8 differs from his
equation from glucose to lactate is without grounds.
Mitchell misunderstands our statement that 'no net
consumption or production occurs in glycolysis' unless he
accepts the dual role of inorganic phosphate, one of which
is that acting partly as a water molecule. This dual role of
phosphate is the essence of our theory.
Mitchell's bringing up of arsenate as the substitute for
Pi only dilutes the argument, since the role of arsenate is
mechanistically different from that of P~ and the interpretation for the stoichiometry requires mechanistic consideration as stated above.
Mitchell's final equation for the overall reaction for the
complete biological oxidation of glucose is indeed our
starting point for the deliberation of the glucose oxidation
(eqn 1).
C6H1206~- 602 -4-36ADP + 2GDP + 38Pi
~6CO2 + 36ATP + 2GTP + 44H20
(1)
There is no problem in this overall equation. We do not
simply discuss the stoichiometric coefficients themselves
in eqn 1. It is when eqn 1 is compared with the combustion
of glucose in a calorimeter (eqn 2) that the problem
arises.
C6H1206 -+-602---* 6 C 0 2 + 6 H 2 0
(2)
In both the biological and nonbiological oxidative degradation of glucose, 602 is consumed per glucose and 6CO2
is produced. However, with respect to H20, there is an
apparent discrepancy between eqns 1 and 2. Namely, as
shown in Table 1 in our paper, and as described by
Mitchell, 44H20 in eqn 1 can be subdivided into 34H20
produced by ATP synthase, 2H20 produced by enolase,
4H20 consumed by the citric acid cycle, and 12H20 pro-
BIOCHEMICAL EDUCATION 24(4) 1996
duced by cytochrome c oxidase (34 + 2 - 4 + 12 = 44). By
subtracting 34H20 involved in the ATP synthase reaction
from 44H20, the net total of 10H20 is produced per each
glucose molecule rather than 6H20 as in eqn 2, the difference being 4H20. So, how can one explain the apparently
inconsistent number of water produced, 6H20 or 10H20?
Thus, Mitchell's claim that 'the overall stoichiometry can
be explained quite readily by summing the individual reactions' cannot be accepted. Is the overall reaction of the
biological oxidation of glucose different from eqn 2 in the
combustion chamber? Should the net total of 6H20 be
produced per glucose in the organisms too? It is our
paper 2 that discusses this apparent discrepancy and
answers the question.
Although the consumption and production reactions of
H20 are individually described in most textbooks, the stoichiometry of'6H20' is not explained, or is only touched on
explicitly or implicitly. In eqn 1, the net total of H20 would
be calculated to be 6H20 per glucose molecule if one
subtracts the total number of 38H20 involved in the ATP/
GTP synthesis reaction (irrespective of whether ATP/
GTP is formed by substrate-level phosphorylation
or by oxidative phosphorylation):
A D P ( G D P ) + P i ~ A T P ( G T P ) + H20,
from the total number of 44H20 produced. This calculation would seem self-evident, probably because the combustion equation of glucose (eqn 2) is only too natural.
However, contrary to oxidative phosphorylation where
one molecule of H20 is produced for each ATP molecule
synthesized, no production of H20 occurs in the substratelevel phosphorylations by phosphoglycerate kinase, pyruvate kinase or succinyl-CoA synthetase. Hence the above
explanation of '6H20' is not justified.
In order to determine the stoichiometry, we need not
consider the reaction mechanisms involved) On the other
hand, it does not suffice to merely compare the numbers
of each element before and after each reaction, as pointed
out in the Footnotes 24 and 25 of our paper. 2 It is necessary to take into account the mechanisms as well for the
better understanding and interpretation of the
stoichiometry.
References
1 Mitchell, R A (1996) Biochem Educ 24, 207-208
2 Horiike, K, Miura, R, Ishida, T and Nozaki, M (1996) Biochem Educ 24,
17-20