J Mol Evol (1996) 43:293–303 © Springer-Verlag New York Inc. 1996 The Puzzle of the Krebs Citric Acid Cycle: Assembling the Pieces of Chemically Feasible Reactions, and Opportunism in the Design of Metabolic Pathways During Evolution Enrique Mele´ndez-Hevia,1 Thomas G. Waddell,2 Marta Cascante3 1 Departamento de Bioquı´mica, Facultad de Biologı´a, Universidad de La Laguna, 38206 Tenerife, Canary Islands, Spain 2Department of Chemistry, University of Tennessee at Chattanooga, 615 McCallie Avenue, Chattanooga, TN 37403, USA 3 Departamento de Bioquı´mica, Facultad de Quı´mica, Universidad de Barcelona, Martı´ i Franque´s 1, 08028 Barcelona, Spain Received: 10 May 1995 / Accepted: 3 November 1996 Abstract. The evolutionary origin of the Krebs citric cal solutions into a single-solution problem, with the ac- acid cycle has been for a long time a model case in the tual Krebs cycle demonstrated to be the best possible understanding of the origin and evolution of metabolic chemical design. Our results also allow us to derive the pathways: How can the emergence of such a complex rules under which metabolic pathways emerged during pathway be explained? A number of speculative studies the origin of life. have been carried out that have reached the conclusion that the Krebs cycle evolved from pathways for amino Key words: Krebs cycle — Evolution — Metabolism acid biosynthesis, but many important questions remain — Citric acid cycle — Chemical design open: Why and how did the full pathway emerge from there? Are other alternative routes for the same purpose possible? Are they better or worse? Have they had any Introduction opportunity to be developed in cellular metabolism evo- lution? We have analyzed the Krebs cycle as a problem During the origin and evolution of metabolism, in the of chemical design to oxidize acetate yielding reduction first cells, when a need arises for a new pathway, there equivalents to the respiratory chain to make ATP. Our are two different possible strategies available to achieve analysis demonstrates that although there are several dif- this purpose: (1) create new pathways utilizing new com- ferent chemical solutions to this problem, the design of pounds not previously available or (2) adapt and make this metabolic pathway as it occurs in living cells is the good use of the enzymes catalyzing reactions already best chemical solution: It has the least possible number existing in the cell. Clearly, the opportunism of the sec- of steps and it also has the greatest ATP yielding. Study ond strategy, when it is possible, has a number of selec- of the evolutionary possibilities of each one—taking the tive advantages, because it allows a quick and economic available material to build new pathways—demonstrates solution of new problems. Thus, in the evolution of a that the emergence of the Krebs cycle has been a typical new metabolic pathway, new mechanisms must be cre- case of opportunism in molecular evolution. Our analysis ated only if ‘‘pieces’’ to the complete puzzle are missing. proves, therefore, that the role of opportunism in evolu- Creation of the full pathway by a de novo method is tion has converted a problem of several possible chemi- expensive in material, time-consuming, and cannot com- pete with the opportunistic strategy, if it can achieve the new specific purpose. We demonstrate here the oppor- Abbreviations: pyr: pyruvate; OAA: oxaloacetate tunistic evolution of the Krebs cycle reorganizing and Correspondence to: E. Mele´ndez-Hevia assembling preexisting organic chemical reactions. This 294 idea has been proposed by several groups, but our ap- Mustafa 1972; Kay and Weitzman 1987; Mason 1992; proach to study these problems—mathematical, logical Mortlock 1992; Thauer 1988). reasoning based on bio-organic chemistry mecha- The aim of this work is to investigate the mechanism nisms—has given us many more details of the process used to organize the structure of the Krebs cycle, ana- and has allowed us to derive some new general proper- lyzing all chemical solutions theoretically possible and ties of metabolic evolution. checking whether each of them is biologically feasible. Once the design of a new metabolic sequence is Our results give a clear and illuminating view of cellular achieved, a refinement of the pathway may be necessary, organization; it is the result of an evolutionary history and then, a further optimization process will move the where opportunism has played a very important role. design toward maximum efficiency by reaching optimal A general consideration of the chemical problem pre- values of rate and affinity constants of enzymes. Such an sented by the Krebs cycle leads us to see, in principle, optimization process as a result of natural selection is three basic ways to solve it: (1) direct oxidation of ac- also a well-documented feature of biological evolution. etate through a sequential pathway; (2) oxidation of ac- Previous results from our group on evolutionary optimi- etate by means of a cyclic pathway; and (3) any combi- zation of metabolic design (Mele´ndez-Hevia 1990; Me- nation of these, i.e., acetate is converted into another le´ndez-Hevia and Isidoro 1985; Mele´ndez-Hevia and compound by means of a sequential or cyclic pathway, Torres 1988; Mele´ndez-Hevia et al. 1994) demonstrated and such a compound is then oxidized to CO2 by means that the design of the pentose phosphate and Calvin of another cyclic or sequential pathway. cycles can be mathematically derived by applying opti- mization principles under a well-established physiologi- Linear Pathway cal function. The same was also demonstrated for the Baldwin and Krebs (1981) discarded this first mecha- structure of the glycogen molecule by Mele´ndez-Hevia nism, arguing that a direct oxidation of acetate—without et al. (1993). In the present paper we report our results any cyclic pathway design—would involve the oxidation concerning the Krebs citric acid cycle, defining the rea- of the methyl group, which could not be done by a sons for its particular structure. These results allow us to NAD(P)+ or FAD-dependent dehydrogenase. We know understand the chemical rules which were followed dur- now that this is indeed possible as the methyl group can ing the evolution of metabolic pathways. be transferred to tetrahydrofolate or an equivalent pteri- dine coenzyme and then be oxidized by NAD(P)+ to CO2; such a pathway, shown in Scheme 1, has been Results found in the anaerobic bacterium Desulfotomaculum by Schauder et al. (1986); see also Sporman and Thauer (1988). The Problem of the Krebs Citric Acid Cycle By considering the first stages in the history of life, we may attempt to determine logically under what condi- tions the Krebs cycle was organized and what its first purpose was: (1) In a first stage, the minimal metabolism that had to work in the first protocells was glycolysis, the anaerobic source of ATP; the pentose cycle, to connect the two kinds of sugars (hexose and pentoses); the path- ways for synthesis of all amino acids, nitrogen bases, and some coenzymes; and fatty acid synthesis, necessary to Scheme 1 make membranes. No pathway for amino acid and nitro- gen-base degradation may have existed, because the se- lective value of a mechanism to eliminate organic mate- Cyclic Pathway rial hardly built was not obvious at all. Oxidative and This involves the condensation of acetyl with a reductive reactions within this primitive set of metabolic feeder; therefore, we must analyze the possibilities for pathways make a balanced anaerobiosis possible, as has the feeder structure and the sequence of possible reac- been shown by Hochachka and Mustafa (1972). (2) At a tions that could recover it. Although there are some im- second stage, the respiratory chain is organized— portant chemical constraints for this, a number of possi- although not necessarily with oxygen as electron accep- bilities exist, since either the methyl group or the car- tor—involving the pathway for the heme-group synthe- bonyl of the acetyl could intervene, and several different sis; then, such a new source of ATP could allow the compounds could be involved in the reactions in each development of gluconeogenesis. (3) In a third stage, the case. For example, if condensation is made through the whole pathway of the Krebs cycle was organized. There methyl group of the acetyl, then either addition to a car- is a general agreement on this view (see Hochachka and bonyl group of the feeder or Michael addition to an 295 Table 1. All possible cases for reaction between the acetyl and the occur in some metabolic reactions, but imines are very feeder to give a cyclic pathway for acetyl oxidation unstable to hydrolysis, and, thus, inappropriate as a feeder. For the case (a2) the carbon which accepts the a. Attack of the methyl of acetyl on the feeder (acetyl needs a carbanion activator) methyl attack must be bonded to a leaving group, such as a.1. Addition to a double bond a phosphate, or a thio-derivative. a11. to C4O a12. to C4C (enone) a2. Substitution (a leaving group is necessary) Case a11 (Addition to a Double Bond C=O). Here the b. Attack of the feeder on the carbonyl of acetyl carbonyl group of the feeder could be a ketone, aldehyde (feeder needs a carbanion activator) or carboxyl group, and the feeder can also have a vari- b.1 Feeder attacks with a methyl able number of carbons. In each case, a different se- b.2 Feeder attacks with a methylene quence of reactions is produced, but the problem we are considering is whether some of these chains can lead to the feeder again making the cyclic pathway possible. The enone could take place.