Portal de Periódicos da CAPES

Evolution of Amino Acid Metabolism Inferred through Cladistic Analysis

2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês

10.1074/jbc.m213028200

1083-351X

Chomin Cunchillos, Guillaume Lecointre,

Origins and Evolution of Life

Because free amino acids were most probably available in primitive abiotic environments, their metabolism is likely to have provided some of the very first metabolic pathways of life. What were the first enzymatic reactions to emerge? A cladistic analysis of metabolic pathways of the 16 aliphatic amino acids and 2 portions of the Krebs cycle was performed using four criteria of homology. The analysis is not based on sequence comparisons but, rather, on coding similarities in enzyme properties. The properties used are shared specific enzymatic activity, shared enzymatic function without substrate specificity, shared coenzymes, and shared functional family. The tree shows that the earliest pathways to emerge are not portions of the Krebs cycle but metabolisms of aspartate, asparagine, glutamate, and glutamine. The views of Horowitz (Horowitz, N. H. (1945) Proc. Natl. Acad. Sci. U. S. A. 31, 153–157) and Cordón (Cordón, F. (1990) Tratado Evolucionista de Biologia, Aguilar, Madrid, Spain), according to which the upstream reactions in the catabolic pathways and the downstream reactions in the anabolic pathways are the earliest in evolution, are globally corroborated; however, with some exceptions. These are due to later opportunistic connections of pathways (actually already suggested by these authors). Earliest enzymatic functions are mostly catabolic; they were deaminations, transaminations, and decarboxylations. From the consensus tree we extracted four time spans for amino acid metabolism development. For some amino acids catabolism and biosynthesis occurred at the same time (Asp, Glu, Lys, Leu, Ala, Val, Ile, Pro, Arg). For others ultimate reactions that use amino acids as a substrate or as a product are distinct in time, with catabolism preceding anabolism for Asn, Gln, and Cys and anabolism preceding catabolism for Ser, Met, and Thr. Cladistic analysis of the structure of biochemical pathways makes hypotheses in biochemical evolution explicit and parsimonious. Because free amino acids were most probably available in primitive abiotic environments, their metabolism is likely to have provided some of the very first metabolic pathways of life. What were the first enzymatic reactions to emerge? A cladistic analysis of metabolic pathways of the 16 aliphatic amino acids and 2 portions of the Krebs cycle was performed using four criteria of homology. The analysis is not based on sequence comparisons but, rather, on coding similarities in enzyme properties. The properties used are shared specific enzymatic activity, shared enzymatic function without substrate specificity, shared coenzymes, and shared functional family. The tree shows that the earliest pathways to emerge are not portions of the Krebs cycle but metabolisms of aspartate, asparagine, glutamate, and glutamine. The views of Horowitz (Horowitz, N. H. (1945) Proc. Natl. Acad. Sci. U. S. A. 31, 153–157) and Cordón (Cordón, F. (1990) Tratado Evolucionista de Biologia, Aguilar, Madrid, Spain), according to which the upstream reactions in the catabolic pathways and the downstream reactions in the anabolic pathways are the earliest in evolution, are globally corroborated; however, with some exceptions. These are due to later opportunistic connections of pathways (actually already suggested by these authors). Earliest enzymatic functions are mostly catabolic; they were deaminations, transaminations, and decarboxylations. From the consensus tree we extracted four time spans for amino acid metabolism development. For some amino acids catabolism and biosynthesis occurred at the same time (Asp, Glu, Lys, Leu, Ala, Val, Ile, Pro, Arg). For others ultimate reactions that use amino acids as a substrate or as a product are distinct in time, with catabolism preceding anabolism for Asn, Gln, and Cys and anabolism preceding catabolism for Ser, Met, and Thr. Cladistic analysis of the structure of biochemical pathways makes hypotheses in biochemical evolution explicit and parsimonious. Cellular metabolism is a complex process involving about a thousand chemical reactions catalyzed by globular proteins, enzymes. As any other biological phenomenon, metabolism has a structure that is the product of an evolutionary history. How can we infer that history? Because the history and interrelationships of living organisms is based on comparative anatomy, the history of metabolism must be reconstructed by the comparative analysis of the structure of its components (1Schoffeniels E. Biochimie Comparée. Masson, Paris1984Google Scholar, 2Cordón F. Tratado Evolucionista de Biologia. Aguilar, Madrid, Spain1990Google Scholar, 3Cunchillos C. Tort P. Pour Darwin. Presses Universitaires de France, Paris1997: 425-447Google Scholar, 4Michal G. Biochemical Pathways. John Wiley & Sons, Inc., New York1999Google Scholar). Biochemists recognized this necessity long ago but have never used comparative methods that formally controlled the consistency of biochemical evolutionary theories (3Cunchillos C. Tort P. Pour Darwin. Presses Universitaires de France, Paris1997: 425-447Google Scholar, 4Michal G. Biochemical Pathways. John Wiley & Sons, Inc., New York1999Google Scholar, 5Jensen R.A. Annu. Rev. Microbiol. 1976; 30: 409-425Crossref PubMed Scopus (810) Google Scholar, 6Cavalier-Smith T. Cold Spring Harbor Symp. Quant. Biol. 1987; 52: 805-824Crossref PubMed Scopus (80) Google Scholar, 7Meléndez-Hevia E. Waddell T.G. Cascante M. J. Mol. Evol. 1996; 43: 293-303Crossref PubMed Scopus (127) Google Scholar, 8Martin W. Müller M. Nature. 1998; 392: 37-41Crossref PubMed Scopus (883) Google Scholar). In systematics, the science of classification of living things, there are now standardized comparative methods to infer events of the past with a measurement of the consistency of a theory. The standard comparative method starts with a number of things to compare. These are known as "operational taxonomic units," "terminals," often "species," or "individuals." These words are not strictly equivalent. By convenience we often call them "taxons." Among those different things we detect that some structures are under different versions. To formalize that observation we create a column in a table called a "matrix" in which a given version is associated with 0, and the other version is associated with 1 in the list of things to compare (versions can be also "coded" 2, 3, etc. depending on the number found). The column is a character; its version as 0 or 1 is the character state. We bet that the versions are homologous; sameness must come from common ancestry. That bet is called "primary homology." Once the matrix is filled with all the characters we were able to detect for that collection of things, a phylogenetic tree is retained according to a criterion explained below. Onto that tree changes of character states become hypothetical events of transformation, and the tree yields a relative order of these events. That tree will show for each character whether that bet is won or lost. If a state appears only once in the tree, the bet is won, and it is called a secondary homology, or a synapomorphy; the state is present from common ancestry in those things that have it. If a state of a character is associated with more than one event, the bet is lost. It is homoplasy, i.e. sameness without common ancestry. But how do we choose a tree over others? For a given number of taxons there are a limited but high number of possible trees, which are different theories of interrelationships. In science theories are to be compared in terms of internal consistency. The theory that is the most consistent is the one that appeals the less ad hoc hypotheses. The most parsimonious tree is the best theory (against alternative trees) because it requires the smallest number of ad hoc hypotheses of transformation onto its branches. This is the reason why one always associates to the most parsimonious tree the number of transformations ("number of steps") and measurements of internal consistency, like the consistency index (C.I.) 1The abbreviations used are: C.I.consistency indexR.I.retention indexKCKrebs cyclePLPpyridoxal phosphate. and the retention index (R.I.). We propose the use of such a parsimony analysis (9Hennig W. Grundzüge einer Theorie der Phylogenetischen Systematik. Deutscher Zentralverlag, Berlin1950Google Scholar, 10Hennig W. Phylogenetic Systematics. University of Illinois Press, Urbana and Chicago, IL1966Google Scholar, 11Farris J.S. Platnick N. Funk V A. Advances in Cladistics. Vol. 2. Columbia University Press, New York1983: 1-36Google Scholar, 12Forey P.L. Humphries C.J. Kitching I.L. Scotland R.W. Siebert D.J. Williams D.M. Cladistics: A Practical Course in Systematics. Clarendon Press, Oxford1992Google Scholar, 13Darlu P. Tassy P. Reconstruction Phylogénétique: Concepts et Méthodes. Masson, Paris1993Google Scholar) to infer the relative timing of emergence of a number of metabolic pathways, i.e. aliphatic amino acid metabolism and portions of the Krebs cycle, in order to shed light on the earliest pathways and enzymatic functions among them. consistency index retention index Krebs cycle pyridoxal phosphate. In 1945 Horowitz (14Horowitz N.H. Proc. Natl. Acad. Sci. U. S. A. 1945; 31: 153-157Crossref PubMed Google Scholar) postulated that the earliest biosynthetic pathways evolved in a backward direction if life began in a rich soup of organic molecules. If primitive cells were using a particular external nutrient, soon this organic molecule would be depleted in the environment. A selective advantage could be obtained by organisms able to synthesize this nutrient from an available precursor. Each biosynthetic step was selected according to successive depletions of precursors in the environment. The first enzyme to appear in the biosynthetic pathway was, therefore, the most distal (i.e. downstream) in the pathway. Confluence of pathways was selected because it saved energy. This energy is used for other needs that will be more difficult to satisfy for competitor cells without confluence. This optimization of pathways is considered as a general basic rule of comparative biochemistry (1Schoffeniels E. Biochimie Comparée. Masson, Paris1984Google Scholar). For these early anabolisms, common enzymes or common reactions shared by two (or more) synthetic pathways are distal and are, therefore, evidence for common ancestry for these pathways. In terms of kinship pathways sharing these enzymes are judged to be closer to each other than to other pathways not using these enzymes. In 1990, Cordón (2Cordón F. Tratado Evolucionista de Biologia. Aguilar, Madrid, Spain1990Google Scholar) proposed a symmetrical scenario of catabolic pathways. Early forms of life extracted energy from the degradation of substrates available in the environment into a product. Selective advantage was obtained for those able to produce a supplementary reaction of deeper degradation of this product, therefore obtaining more energy from the original substrate. Confluence is selected by obtaining the transformation of another substrate into an intermediate product already present in the protocell. The first reactions to appear in evolution of catabolism are proximal ones (i.e. upstream ones). The common distal elongation of two branched catabolic pathways is, therefore, a phenomenon whose final result will be evidence for common ancestry of these pathways. Two catabolic pathways sharing one or several distal portions of catabolism are supposed to be more closely related to each other than to other pathways. But there is a risk, which consists of the late connection of an "opportunistic" catabolic pathway once the early catabolism onto which it connects is already complete. In that case the common downstream portion is not evidence for common ancestry but just convergence obtained by recruitment, a phenomenon recognized for having played a role in biochemical evolution (5Jensen R.A. Annu. Rev. Microbiol. 1976; 30: 409-425Crossref PubMed Scopus (810) Google Scholar, 15Petsko P. Kenyon G.L. Gerlt J.A. Ringe D. Kozarich J.W. Trends Biochem. Sci. 1993; 18: 372-376Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 16Copley S.D. Trends Biochem. Sci. 2000; 25: 261-265Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). It is sameness without common ancestry. When we reconstruct the past, homoplasy appears when there are character conflicts due to such similarities obtained by evolutionary convergence or reversion. The risk of homoplasy in our data just depends on the relative timing between events of distal elongation of a pathway and the connecting event of another pathway. An early connecting event followed by distal elongation will provide good phylogenetic indicators. A late connecting event (late in time and/or late in the pathway) will probably bring homoplasy. As in any other study of systematics where putative homologies are coded into a matrix, there are risks of homoplasy to carry on. We make the bet that this homoplasy will not swamp the phylogenetic signal. According to Cordón (2Cordón F. Tratado Evolucionista de Biologia. Aguilar, Madrid, Spain1990Google Scholar) the symmetrical scenario is not rigid and can change from one type of metabolites to another; the order of development of a given pathway depends on the position and availability of the initial substrate and/or final product. The above timings in the genesis of reactions in anabolic pathways and catabolic pathways are valid because the final product and the initial substrate, respectively, are imposed from the outside to the cell, at least initially. Alternative scenarios can be obtained for transformations starting from products already integrated into the cellular metabolism. New biosynthetic pathways can develop in a forward direction by the addition of new enzymes and reactions to pre-existing pathways. For example, the urea cycle uses the biosynthesis of arginine (17Takiguchi M. Matsubasa T. Amaya Y. Mori M. BioEssays. 1989; 10: 163-166Crossref PubMed Scopus (40) Google Scholar). Thus, Cordón (2Cordón F. Tratado Evolucionista de Biologia. Aguilar, Madrid, Spain1990Google Scholar) proposed a forward development for amino acid catabolism, fatty acids anabolism, and glycogenesis and a backward development for amino acid anabolism, fatty acids catabolism, and glycolysis (confirmed in 1993 by Fothergill-Gilmore and Michels (18Fothergill-Gilmore L.A. Michels P.A.M. Prog. Biophys. Mol. Biol. 1993; 59: 105-235Crossref PubMed Scopus (378) Google Scholar)). However, late opportunistic connections of pathways can complicate the general ordering of metabolism development as predicted by Cordón (2Cordón F. Tratado Evolucionista de Biologia. Aguilar, Madrid, Spain1990Google Scholar). For example, some catabolic pathways can develop backwards by opportunistic connecting to intermediate compounds from already developed pathways (e.g. Cordón's catabolism of serine). For the same reasons some anabolic pathways can develop forward (e.g. Cordón's anabolism of methionine and threonine). The complexity of the matter is increased by the fact that some biochemical pathways can be considered both catabolic and anabolic (pentose phosphate shunt, glycolysis, gluconeogenesis, the Krebs Cycle). Therefore, it is clear for all biochemists today that there is no direct link between being either anabolic or catabolic and having grown either forward or backward. This is one of the reasons why it is important to start the present methodological improvement with simple examples, like amino acids, for which in most cases the catabolic pathway is different from the anabolic pathway. The evolutionary development of pathways has to do with the darwinian concept of descent with modification, justifying the use of cladistic analysis (19Kluge A.G. Cladistics. 2001; 17: 199-210Crossref Google Scholar) for metabolic pathways. By using that concept we bet that present similarities in pathways detected through shared enzymes and enzymatic reactions can be interpreted as the result of pathway transformations through time. Comparison of pathways can, therefore, be followed by phylogenetic reconstruction of metabolic pathways. Why focus on amino acids and not on nucleic acids, fatty acids, or monosaccharides? The reasons are manyfold. Amino acids are among the earliest abiotic sources of energy and molecules for protocells without excluding other possible sources (for instance, sunlight for energy, pentoses). Consequently, their metabolism must have been involved among the earliest biochemical reactions. Simple amino acids have been obtained experimentally in an abiotic way for example by Miller in 1953 (20Miller S.A. Science. 1953; 117: 528Crossref PubMed Scopus (2012) Google Scholar), who obtained glycine, alanine, glutamate, and aspartate from simple molecules like ammoniac, hydrogen, methane, and water. Serine can be obtained abiotically in the presence of formaldehyde (20Miller S.A. Science. 1953; 117: 528Crossref PubMed Scopus (2012) Google Scholar). Even if the chemical environment of the primitive soup is today interpreted in a different manner than it was at the time of Miller, free amino acids are still found in abiotic and extraterrestrial environments, for example, in meteorites, like the famous carbonaceous chondrites, which are probably the source on primitive earth. Moreover, among classical candidates for early energy providers (fatty acids, monosaccharides, amino acids), amino acids are the only chemical precursors whose structure is complex enough to contain all the atoms and reactive groups necessary for most of the reactions necessary for central metabolism. Other compounds like monosaccharides, polysaccharides, and fatty acids have a poorer variety of atoms and groups and are rather monotonous. However, the strongest argument for early availability of amino acids to protocells might be the fact that any metabolism defined as a coordinated network of enzymatic activities performed by proteins needs in its very early steps amino acid anabolism and catabolism for these proteins. Amino acid metabolism might, therefore, have preceded any other metabolism, even the most central metabolism like the Krebs cycle. However, this question is still debated. Whatever the metabolic specialization found in diverse living organisms (heterotrophy, photosynthetic autotrophy, chemosynthetic autotrophy, various forms of respiration and fermentation), there is a universal core of about 50 metabolic pathways involving the anabolism and catabolism of amino acids, fatty acids, saccharides (the glycolysis and the glycogenesis, the pentose phosphate pathway), and the Krebs cycle. Because the Krebs cycle is the point of confluence of all other metabolic pathways, it is sometimes viewed as primitive. That view is, however, challenged by several lines of evidence. Molecules entering the Krebs cycle (oxo acids and acyl-CoAs) are intermediate metabolites that are diversely interpreted with regard to their availability in primitive abiotic environments. Some biochemists consider them as most likely the products of a peripheral cellular metabolism. For example, the origin of the Krebs cycle was thought to be secondary and composite by Schoffeniels (1Schoffeniels E. Biochimie Comparée. Masson, Paris1984Google Scholar, 21Schoffeniels E. Les Cahiers de Biochimie (Paris). Vaillant-Carmanne Maloine, Paris1981Google Scholar), Gest (22Gest H. FEMS Microbiol. Lett. 1981; 12: 209-215Crossref Scopus (24) Google Scholar, 23Gest H. Biochem. Soc. Symp. 1987; 54: 3-16PubMed Google Scholar), and Meléndez-Hevia et al. (7Meléndez-Hevia E. Waddell T.G. Cascante M. J. Mol. Evol. 1996; 43: 293-303Crossref PubMed Scopus (127) Google Scholar). Others consider them as most likely available right from the beginning; for instance, carbon dioxide, water, and sunlight provide oxalic acid through pyrite catalysis. Then, the primitive soup would have been rich in dicarboxylic acids. These debates led us to incorporate two portions of the Krebs cycle as taxons in the matrix along with the metabolic pathways of each of the 16 aliphatic amino acids in our data sets. By analyzing anabolism and catabolism all together following the protocol of Cunchillos and Lecointre (24Cunchillos C. Lecointre G. Barriel, V. Bourgoin T. Biosystema 18. Société Française de Systématique, Paris2000: 87-106Google Scholar, 25Cunchillos C. Lecointre G. C. R. Biologies. 2002; 325: 119-129Crossref PubMed Scopus (10) Google Scholar) for catabolism only, we compared portions of the Krebs cycle both to amino acid catabolism and anabolism. The ultimate question was to find the earliest pathways and enzymatic functions among peripheral amino acid metabolisms and portions of the Krebs cycle. This work is a cladistic analysis of the structure of a part of the cellular metabolism. This is allowed by taking each pathway as a taxon and shared enzymes, shared enzymatic functions, and shared cofactors as characters. Each taxon starts from the initial substrate and includes the pathway until its entry into the Krebs cycle or starts from the metabolites of the Krebs cycle and includes the pathway to the amino acid. For example, the taxon dASN1 is the catabolic pathway from asparagine to oxaloacetate, and the enzymes and functions along this pathway will be the characters of this taxon (Fig. 1, characters 2, 4, 11, 13, and 28). The taxon sASN1 is the anabolic pathway from oxaloacetate to asparagine (Fig. 1, characters 2, 4, 11, 28, 56, 106, and 107). In this example the pathways used for the degradation ("d") and the synthesis ("s") of the amino acid are very similar; however, this is not the case for all taxons. Some amino acids can be degraded or synthesized through several possible ways (cysteine, aspartate, asparagine, glutamate, glutamine, threonine). In these cases each way is taken separately as an additional taxon (for instance, dCYS1, dCYS2, dCYS3 for the degradation (d) of cysteine (Cys) after the pathways 1, 2, and 3, respectively). The Krebs cycle (KC) is considered in two portions, each beginning with an oxo acid, which is a point of entrance into the cycle and also a point of output. These two oxo acids are also, among the metabolites of the Krebs cycle, the closest to amino acids structurally speaking. The portion KC1 begins with oxaloacetate and ends with α-oxoglutarate; the portion KC2 begins with α-oxoglutarate and ends with oxaloacetate. Aromatic amino acids have not been considered at this stage of cellular evolution because their complex metabolism needs too much oxygen and is only possible once the metabolism of aliphatic amino acids is set.Fig. 2List of characters of the matrix (Fig. 1). Each number and name associated corresponds to a column in the matrix, with the corresponding number of international nomenclature (Enzyme Nomenclature) and homology types defined in the text. For example, the 26th character is the alanine aminotransferase; it corresponds to the 26th column in Fig. 1, the international nomenclature is 2.6.1.2, and in the text it is a primary homology of type I.View Large Image Figure ViewerDownload (PPT) Our aim is first to understand phylogenetic interrelationships of aliphatic amino acid catabolic and anabolic pathways, second to discover in the most parsimonious tree the earliest metabolic pathway, and third to discover the first enzymatic functions associated to them. This should answer the following connected questions; Is the Krebs cycle the first? Does amino acid catabolism appear before amino acid anabolism? Can we test the expected order of metabolism development as predicted by Horowitz (14Horowitz N.H. Proc. Natl. Acad. Sci. U. S. A. 1945; 31: 153-157Crossref PubMed Google Scholar) and Cordón (2Cordón F. Tratado Evolucionista de Biologia. Aguilar, Madrid, Spain1990Google Scholar)? In other words, does anabolism develop backwards and catabolism forwards? Comparing differences in metabolisms of extant living organisms is of no help in reaching this aim because all the corresponding events are more differences in metabolism regulation than differences in structure of pathways (1Schoffeniels E. Biochimie Comparée. Masson, Paris1984Google Scholar) and, anyway, might have been posterior to the very early events we intend to infer. Comparing semantids of Zuckerkandl and Pauling (26Zuckerkandl E. Pauling L. J. Theor. Biol. 1965; 8: 357-366Crossref PubMed Scopus (764) Google Scholar), i.e. DNA sequences or protein sequences, is of no help because it leads to severe problems of linear sequence homology and would infer mutational changes that also are posterior to the occurrence of these early metabolisms. Problems with sequence data are even more complex. The present taxons are pathways. Each 0 and 1 coded into a cell of the matrix could have been replaced by the sequence of the corresponding enzyme in a given model organism. Then, comparing sequences would have mixed up different gene duplication histories, leading to no trees at all. Moreover, we have no theoretical model that links ordering events of gene duplication within a putative primitive genome with ordering the rise and organization of enzymatic activities in a protocellular metabolism. At last, the present work infers nothing about information storage, replication, and the RNA world. If shared enzymes or similar enzymes are evidence for common ancestry of metabolic pathways, similarities in the structure of active sites would be sufficient to formulate putative homologies. However, only a few active sites (27Knowles J.R. Nature. 1991; 350: 121-124Crossref PubMed Scopus (494) Google Scholar) are known in detail compared with the large number of known enzyme types (4Michal G. Biochemical Pathways. John Wiley & Sons, Inc., New York1999Google Scholar). We are, therefore, led to consider similarities in catalytic reactions and enzymatic mechanisms as reflecting similarities in active sites. The higher the specificity, the more accurate is this reflection. In the same way, considering the generally accepted idea that enzymes evolved from low specificities to high specificities (5Jensen R.A. Annu. Rev. Microbiol. 1976; 30: 409-425Crossref PubMed Scopus (810) Google Scholar, 15Petsko P. Kenyon G.L. Gerlt J.A. Ringe D. Kozarich J.W. Trends Biochem. Sci. 1993; 18: 372-376Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 28Holden J.T. J. Theor. Biol. 1968; 21: 97-102Crossref PubMed Scopus (6) Google Scholar, 29Jallon J.M. Hervé G. L'Evolution des Protéines. Masson, Paris1983: 92-103Google Scholar, 30Pierard A. Hervé G. L'Evolution des Protéines. Masson, Paris1983: 53-66Google Scholar, 31O'Brien P.J. Herschlag D. Chem. Biol. (Lond.). 1999; 6: 91-105Abstract Full Text PDF PubMed Scopus (618) Google Scholar), putative common ancestry of pathways can be postulated not only on the basis of shared enzymes with high specificities but also on the basis of very similar reactions. Similarity in function must correspond to an underlying similarity in structure of active sites, a structural similarity that comes from common ancestry. Recognizing that two metabolic pathways share the same reaction with high specificity for substrate uses a strict criterion of primary homology (32de Pinna M.C.C. Cladistics. 1991; 7: 367-394Crossref Scopus (817) Google Scholar), whereas recognizing a common family of reaction relaxes this criterion with a risk of homoplasy obtained by convergence or recruitment. There is no reason for considering that this risk is higher in the present case than in aligned DNA sequences or in classical morpho-anatomical matrices, both usual in systematics (33Sanderson M.J. Hufford H. Homoplasy. Academic Press, Inc., New York1996Google Scholar). The criteria of primary homology is 4-fold: shared specific enzymatic activity (I), shared enzymatic function without shared specificity for substrate (IIa), shared coenzymes (IIb), and shared family of function (IIc). Several pathways share the same enzyme with high specificity for its substrate. The enzyme itself is the hypothesis of primary homology. For reversible enzymes this is valid either for a given reaction or the reverse reaction. The absence is coded 0, and presence is coded 1. For instance, the catabolism of aspartate and asparagine both use the aspartate aminotransferase, which transforms aspartate into oxaloacetate. The anabolism of these two amino acids use the same enzyme for the reverse reaction, which transforms oxaloacetate into aspartate. The character is called "aspartate aminotransferase" (character 12); it is coded 1 for taxons dASP2, dASN2, sASP2, and sASN2 and 0 for dGLU, sGLU1, dALA, and sALA, for example. This type of homology is used in characters 11–26, 29–105, 109, and 110. IIa; Shared Enzymatic Functions—Several pathways utilize the same enzymatic functions, i.e. exhibit the same kind of chemical transformation, without considering the specificity of each enzyme for its substrate. The underlying hypothesis is that similarity in enzymatic function must correspond to similarity in the structure of active sites, with the hypothesis that enzymes must have evolved from generalist active sites to specialized ones (28Holden J.T. J. Theor. Biol. 1968; 21: 97-102Crossref PubMed Scopus (6) Google Scholar, 29Jallon J.M. Hervé G. L'Evolution des Protéines. Masson, Paris1983: 92-103Google Scholar, 30Pierard A. Hervé G. L'Evolution des Protéines. Masson, Paris1983: 53-66Google Scholar, 31O'Brien P.J. Herschlag D. Chem. Biol. (Lond.). 1999; 6: 91-105Abstract Full Text PDF PubMed Scopus (618) Google Scholar). When the substrate is present,