How the Citric Acid Cycle Was Discovered The detailed biochemistry of the
citric acid cycle was determined by several researchers over a period of
decades. In a 1937 article, Krebs and Johnson summarized their work and the
work of others in the first published description of this pathway.
The methods used by these researchers were very different from those of modern
biochemistry. Radioactive tracers were not commonly available until the 1940
s, so Krebs and other researchers had to use nontracer techniques to work out
the pathway. Using freshly prepared samples of pigeon breast muscle, they
determined oxygen consumption by suspending minced muscle in buffer in a
sealed flask and measuring the volume (in \(\mu \mathrm{L}\) ) of oxygen
consumed under different conditions. They measured levels of substrates
(intermediates) by treating samples with acid to remove contaminating
proteins, then assaying the quantities of various small organic molecules. The
two key observations that led Krebs and colleagues to propose a citric acid
cycle as opposed to a linear pathway (like that of glycolysis) were made in
the following experiments.
Experiment I: They incubated \(460 \mathrm{mg}\) of minced muscle in 3
\(\mathrm{mL}\) of buffer at \(40^{\circ} \mathrm{C}\) for 150 minutes. Addition
of citrate increased \(\mathrm{O}_{2}\) consumption by \(893 \mu \mathrm{L}\)
compared with samples without added citrate. They calculated, based on the
\(\mathrm{O}_{2}\) consumed during respiration of other carbon-containing
compounds, that the expected \(\mathrm{O}_{2}\) consumption for complete
respiration of this quantity of citrate was only \(302 \mu \mathrm{L}\).
Experiment II: They measured \(\mathrm{O}_{2}\) consumption by \(460 \mathrm{mg}\)
of minced muscle in \(3 \mathrm{~mL}\) of buffer when incubated with citrate
and/or with 1-phosphoglycerol (glycerol 1-phosphate; this was known to be
readily oxidized by cellular respiration) at \(40^{\circ} \mathrm{C}\) for 140
minutes. The results are shown in the table.
\begin{tabular}{llc}
1 & No extra & 342 \\
\hline 2 & \(0.3 \mathrm{~mL} 0.2 \mathrm{M}\) 1-phosphoglycerol & 757 \\
\hline 3 & \(0.15 \mathrm{~mL} 0.02 \mathrm{M}\) citrate & 431 \\
\hline 4 & \(0.3 \mathrm{~mL} 0.2 \mathrm{M}\) 1-phosphoglycerol and \(0.15
\mathrm{~mL} 0.02\) & 1,385 \\
& M citrate & \\
\hline
\end{tabular}
a. Why is \(\mathrm{O}_{2}\) consumption a good measure of cellular respiration?
b. Why does sample 1 (unsupplemented muscle tissue) consume some oxygen?
c. Based on the results for samples 2 and 3 , can you conclude that
1-phosphoglycerol and citrate serve as substrates for cellular respiration in
this system? Explain your reasoning.
d. Krebs and colleagues used the results from these experiments to argue that
citrate was "catalytic"that it helped the muscle tissue samples metabolize 1
phosphoglycerol more completely. How would you use their data to make this
argument?
e. Krebs and colleagues further argued that citrate was not simply consumed by
these reactions, but had to be regenerated. Therefore, the reactions had to be
a cycle rather than a linear pathway. How would you make this argument?
Other researchers had found that arsenate \(\left(\mathrm{AsO}_{4}^{3-}\right)\)
inhibits \(a\)-ketoglutarate dehydrogenase and that
malonate inhibits succinate dehydrogenase.
f. Krebs and coworkers found that muscle tissue samples treated with arsenate
and citrate would consume citrate only in the presence of oxygen; under these
conditions, oxygen was consumed. Based on the pathway in Figure 16-7, what was
the citrate converted to in this experiment, and why did the samples consume
oxygen?
In their article, Krebs and Johnson further reported the following: (1) In the
presence of arsenate, \(5.48\) mmol of citrate was converted to \(5.07
\mathrm{mmol}\) of \(a\) ketoglutarate. (2) In the presence of malonate, citrate
was quantitatively converted to large amounts of succinate and small amounts
of \(a\)-ketoglutarate. (3) Addition of oxaloacetate in the absence of oxygen
led to production of a large amount of citrate; the amount was increased if
glucose was also added.
Other workers had found the following pathway in similar muscle tissue
preparations:
Succinate \(\rightarrow\) fumarate \(\rightarrow\) malate \(\rightarrow\)
oxaloacetate \(\longrightarrow \mathrm{p}\)
g. Based only on the data presented in this problem, what is the order of the
intermediates in the citric acid cycle? How does this compare with Figure
16-7? Explain your reasoning.
