Hans Krebs (1900-1981)

Prior to Krebs discovery, experiments by T. Thunberg and F. Batelli and L.S. Stern revealed that minced animal tissues contained substances that could transfer hydrogen atoms from specific intracellular organic acids (including succinate, malate, and citrate) to methylene blue dye, reducing it to a colorless form. Using tissue baths in combination with manometers, a number of scientists discovered that minced tissue suspensions rapidly oxidized citrate, fumarate, malate, and succinate to carbon dioxide in the presence of oxygen.

Albert Szent-Gyorgyi extended these studies by describing a sequence of reactions for succinate oxidation, namely succinate to fumarate to malate to oxaloacetate. He further discovered that adding a small amount of malate or oxaloacetate stimulates the reduction of far more oxygen than is needed to completely oxidize the substance added. He therefore postulated that the addition must trigger oxidization of some endogenous substance in the tissues, perhaps glycogen. Martius and Knoop later discovered another part of the sequence, namely citrate to alpha-ketoglutarate to succinate.

In an elegant series of experiments, Krebs then worked out the cyclic nature of the reactions. He noted that only certain organic acids were readily oxidized by muscle, and found that the oxidation of endogenous carbohydrate or pyruvate could be stimulated by a number of specific acids, all of which turned out to be substrates of the tricarboxylic acid cycle enzymes. Since malonate, which competitively inhibits succinate dehydrogenase, completely stopped the oxidation of pyruvate by the addition of organic acids, he concluded that the succinate to fumarate reaction must be a critical link in a chain of reactions involving all of the known catalytically active acids that can stimulated oxidation of pyruvate.

Krebs discovered the formation of citrate from oxaloacetate and pyruvate, the 'missing link' that allowed the known reactions to form a cyclic sequence. Adding malonate to muscle suspensions caused an accumulation of succinate in the presence of citrate, isocitrate, cis-aconitate, or alpha-ketoglutarate. In the presence of fumarate, malate, or oxaloacetate, succinate also accumulated, clearly establishing a cyclic sequence leading to succinate. Malonate poisoning also limited the ability of oxaloacetate to stimulate the oxidation of pyruvate - where one molecule of oxaloacetate could stimulate the oxidation of many molecules of pyruvate in the uninhibited system, only one molecule of pyruvate was oxidized per molecule of oxaloacetate in the malonate-poisoned system. Thus, pyruvate clearly entered a cyclic system of oxidation of substrates.

It wasn't established until later that citric acid was indeed the first substrate formed from the reaction of pyruvate and oxaloacetate, so the cycle was called simply the tricarboxylic acid cycle for many years. Now, both names are accepted, as well as the term 'Krebs cycle.'

Krebs' own account of the history of the discovery of the cycle can be found in his article, The History of the Tricarboxylic Acid Cycle, Perspect. Biol. Med., 14: 154-170 (1970).