Question

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. $$\text{Unknown environment 'tabular'}$$ 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 $\to$ fumarate $\to$ malate $\to$ oxaloacetate $⟶\mathrm{p}$ 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.

Short answer

Oxygen consumption indicates respiratory substrate use. Both citrate and 1-phosphoglycerol contribute to respiration, but citrate is catalytic, regenerating in a cycle.

Step-by-step solution

Understanding Oxygen Consumption and Respiration

Cellular respiration requires oxygen to produce energy (in the form of ATP) by breaking down substrates like glucose or other organic compounds. The consumption of $ext{O}_2$ is proportional to the energy production, thus making it a reliable indicator of respiratory activity.

Analyzing Baseline Oxygen Consumption

Sample 1 consumes $342$ $ext\mu L$ of oxygen due to inherent metabolic activity in the muscle tissue, where endogenous substrates are oxidized even without external addition.

Evaluating 1-Phosphoglycerol and Citrate as Substrates

In Sample 2, $ext{O}_2$ consumption increases significantly with the addition of 1-phosphoglycerol, indicating it serves as a substrate. Similarly, in Sample 3, citrate causes a smaller but appreciable increase. Thus, both compounds contribute to respiration.

Determining Citrate's Catalytic Role

Samples 2 and 4 show that oxygen consumption is higher in the presence of both 1-phosphoglycerol and citrate compared to either alone. This suggests citrate enhances the oxidation of 1-phosphoglycerol, indicating a catalytic rather than a substrate role.

Arguing for the Regeneration of Citrate

Significant $ext{O}_2$ consumption even after citrate's initial impact suggests its continual presence and regeneration within the cycle, supporting the idea of a cyclic, rather than linear, pathway.

Evaluating Effects of Arsenate and Malonate

In experiments with inhibitors, citrate conversion to other intermediates and continued $ext{O}_2$ consumption despite arsenate or malonate use implies metabolic steps beyond direct citrate oxidation, hinting at its regeneration.

Ordering of Citric Acid Cycle Intermediates

Based on experiments, the pathway is likely: Citrate → α-Ketoglutarate (inhibited by arsenate) → Succinate → Fumarate → Malate → Oxaloacetate (inhibited by malonate). In comparison to Figure 16-7, this order should match well.

Key concepts

History of Biochemistry

Biochemistry has roots reaching back to ancient times, but it emerged as a distinct scientific discipline in the 19th century. Initially, biochemistry focused on studying the chemical processes within living organisms. Over time, this field has advanced dramatically.
One of the most pivotal discoveries was the identification of metabolic pathways. This showed how intricate chemical reactions enable life processes.
The Citric Acid Cycle, also known as the Krebs Cycle, played a crucial role in our understanding of cellular respiration. This pathway illustrates how food is converted into energy within cells and highlights the broader significance of biochemical research in understanding life itself.

Research Methods in Biochemistry

Early biochemistry relied heavily on general chemistry and physics. Researchers used basic equipment like flasks and beakers, which might seem simple by today's standards.
One key method in the early days was indirect observation. Scientists observed how certain elements, like oxygen, were consumed in processes indicative of larger biochemical reactions, especially in studies involving cellular respiration.
Before radioactive tracers, non-radioactive techniques prevailed. Researchers used beautifully simple, yet effective methods, such as measuring oxygen consumption and using acid treatment to separate proteins from other compounds. These approaches were crucial to foundational work, like the discovery of the Citric Acid Cycle.

Metabolic Pathways

Metabolic pathways are sequences of chemical reactions occurring within a cell. These pathways are essential for maintaining life, allowing organisms to grow, reproduce, and respond to their environment.
The Citric Acid Cycle is one of the most significant metabolic pathways. It is a critical component of cellular respiration and plays a vital role in energy production by breaking down molecules to produce ATP, carbon dioxide, and water.

• By understanding these pathways, scientists have been able to develop medications, understand diseases, and enhance agricultural practices.
• Metabolic pathways demonstrate the interconnectedness of biological processes, where the end product of one reaction is typically the starting substance for another.

Cellular Respiration

Cellular respiration converts biochemical energy from nutrients into ATP, and then releases waste products. It is a multi-step process that takes place in the mitochondria of cells.
There are three main stages:

• Glycolysis: The split of glucose into pyruvate, yielding a small amount of ATP.
• The Citric Acid Cycle: Converts acetyl-CoA into carbon dioxide, producing energy-storing molecules like NADH and FADH2.
• Oxidative Phosphorylation: Utilizes the energy-storing molecules to generate a large amount of ATP.

Oxygen is crucial for complete respiration. It acts as the final electron acceptor in the electron transport chain, which is why the consumption of oxygen in experiments can indicate cellular respiration activity.

Krebs Cycle Discovery

The Krebs Cycle discovery was monumental. In 1937, Hans Krebs and William Johnson published their findings on this essential metabolic pathway.
Their work illustrated that the process wasn't linear like glycolysis but cyclical—regenerating its starting material with each turn.
This discovery reshaped how scientists understood metabolism. Using experiments that involved oxygen consumption without modern tools available today, Krebs demonstrated the cycle's catalytic nature.

This landmark discovery showed how cells efficiently extract energy from nutrients, encouraging further research into cellular processes and laying the foundation for modern biochemistry.

Biochemical Experimentation

Biochemical experimentation has evolved significantly from early days. In the 1930s and 40s, scientists, including Krebs, conducted intricate but manual experiments to deduce metabolic pathways.
One typical experiment involved measuring oxygen consumption to infer metabolic activity.
Another involved observing the effect of inhibitors on certain reactions, as they could block specific steps, helping to identify components of metabolic cycles.

• Examples include studying the effects of arsenate and malonate, which inhibit particular enzymes, blocking reactions and suggesting possible intermediate steps in the cycle.
• These early experiments laid the groundwork for complex biochemical techniques we use today, like high-throughput screening and mass spectrometry.