(A summary of Faustino Cordón’s article “The globular proteins. Their supramolecular structure and function”, Mundo Científico, Nº 142, January 1994. pp. 40-47)
Throughout the 19th century, a distinction was made between unorganized ferments (pepsin, amylase, the ferments of the pancreas, emulsin, urease, etc.), molecules whose composition was poorly known that were considered to be related to the vital activity of animals and plants and seemed to act as specific catalysts of simple transformations that can be achieved by other means; and organized ferments, single-cell beings that were also specific producers of alcoholic, lactic and butyric fermentation, etc., which were far more complex processes that could not be reproduced in vitro. A milestone in the hitherto poorly understood differentiation between the two types of ferment (and indeed it marked the development of biochemistry) was reached in 1900, when Buchner discovered that ultrafiltrates of yeast produce the alcoholic fermentation of glucose in the same way as live yeast does. This process was so complex that it took thirty years of biochemical research and led to a series of unexpected and transcendental results:
Eduard Buchner (1860-1917)
1) Alcoholic fermentation (in summary) proved to be a complex process of degradation that consists of ten transformations, each of which requires a specific unorganized ferment: a globular protein.
2) Each of these metabolic transformations occurs in a way that is only conceivable if it is guided by a specific globular protein that is perfectly individualizable and is not the same as a simple molecule (whether or not the molecule is a catalyst).
3) Surprisingly, the process of glycolysis (which is common to countless cells and initiates cellular respiration) consists of the same metabolic transformations as fermentation, and all these transformations are governed by globular proteins, each with specific functions. The later development of biochemistry revealed that the cellular metabolism is a very complex process governed by specific globular proteins and that it is common to all cell types in general and in many particular features, so the diversification results from obvious adaptations of a very old cellular metabolism that subsequently adapted to different trophic environments.
4) Lastly, against the opinion of Claude Bernard that metabolic degradations are a merely chemical process, whereas syntheses require living activity, biochemistry has shown that they are equally complex processes, governed by proteins and obviously coordinated with each other at the service of the cell, in which the metabolism plays a basic function that remains to be interpreted.
The above four points seem to show that the globular protein is the individual agent of sub-cellular life and give us a clear idea of how it acts on the molecules within its scope by governing the intracellular molecules one by one.
The great interest elicited by the discovery of cellular enzymes seems to suggest that the point of view that I am presenting would have been accepted if Berthelot’s conviction that life had to be explained in chemical terms had not taken root. Indeed, Pasteur, who died five years before Buchner’s discovery, firmly challenged this view and considered life to be consubstantial with the cell. History of Biochemistry
One of the key problems for biology (which could place it at the forefront of all the other branches of science) is to resolve the antinomy between the nature of the living being as an integrative unit, which can only be a physical field limited to the units of each level, and the nature of the units of the immediately lower level that form its soma, which are a multiple set that can be very numerous and complex. It seems obvious that the unit can only be understood in terms of the plurality of the soma, and vice versa, and that this interplay can only be understood in the framework of a suitable specific environment of stability.
In the terrestrial biosphere (and in all likelihood in other places of the cosmos), the stratification of living beings into their three biological levels merely culminates the stratification into integrative units of different levels that the experimental sciences have perceived (with difficulty but rigorously) in the inorganic domain. This stratification satisfies the old evidence that animals are as much living units as cells are and also explains why cell somas are, at their level, as complex as animal somas are at their level. Corresponding conclusions are drawn from comparing the globular protein as the unit of a directly supramolecular level with the obvious complexity of its soma, composed specifically of twenty species of molecules (the 20 biological amino acids).
On the basis of this order of ideas, in this work we limit ourselves to selecting a set of specific experimental data of very diverse nature that seem to show, beyond reasonable doubt, that globular proteins are the living beings of the first biological level (the directly supramolecular and infracellular integrative units), whereas nucleic acids are merely associations of molecules produced by the associative activity of specific globular proteins under the control of the cellular action and experience, and to the advantage of the cell.
The data indicating that globular proteins are the integrative units of the first biological level may be divided into several overlapping sets. Here we will discuss only two of them:
1) experimental data proving that the structure of globular proteins corresponds to that of units of a directly supramolecular level; and
2) experimental data proving that only globular proteins act by applying action and experience of directly supramolecular integrative units (analysis of the data on the activity of metabolic proteins).
The following general facts seem to prove that globular proteins have a supramolecular structure.
It is already an indication that they have a size (100,000 daltons) several orders greater than that of the molecules with which they relate (metabolites and coenzymes of 100 daltons).
Differences in size and degree of complexity between enzymes on the one hand and metabolites and coenzymes on the other render unlikely the prevailing idea that, in metabolic transformation, the globular protein with an enzymatic function acts as a simple molecule with respect to the coenzymes and metabolites, which are indeed molecules.
Of course, the supramolecular order of size could be attributed to the fact that globular proteins are a mere association of molecules, just like large nucleic acids (which are even larger than globular proteins in the case of deoxyribonucleic acids). However, in our opinion there are differences between the two types of substance that lead us to think that the molecules of which globular proteins are composed are coordinated in a way that seems suitable for a supramolecular unit (an agent) to be formed on their joint activity.
Let us now consider the structure of molecules that characterizes globular proteins and seems to correspond to the directly supramolecular and infracellular living being.
With respect to the molecular structure of globular proteins, it should be recalled, first, that they all have 20 species of L-α-amino acids (the biological amino acids) linked to each other, with elimination of water, by peptide bonds that tend to form one or more polypeptides per globular protein. Since it was established unequivocally by H.E. Fischer (1852-1919), it has been known that the linear axis of these polypeptides has a highly simple and regular structure.
The molecular structure of globular proteins
Because of their law of formation, these polypeptides grow (and diminish) through their amino and carboxyl groups, which participate in the formation of a chain that has a free amino group at one end and a free carboxyl group at the other, with the residue of each L-α-amino acid placed laterally along the chain. The L-α-amino acids residues have many similarities (order of size, proportions of CH2 groups, etc.) that seem to indicate a common chemical origin, which must have been prior to the formation of the supramolecular globular proteins in the molecular state of biological evolution.
It should be noted that all these L-α-amino acid residues have the same arrangement of esters (except polypeptides with D-α-amino acids in living matter), and this fact cannot be explained by the laws of interaction between molecules.
It is interesting to note, also, that each globular protein -specifically defined by its function in the cell and by the cell of origin- is characterized by a specific sequence of L-α-amino acids in which no regularity can be observed. It therefore seems to be a sequence with no distributive law, but it is the right sequence for the globular protein as a whole to perform a vital function, so it is conserved faithfully from generation to generation of cells for each type of globular protein. All of this seems to indicate that, in the phylogenic refinement of each globular protein, each L-α-amino acid was selected for the place that it occupies because of its contribution to the work of the globular protein in carrying out, in the best way possible, the function that it has had in the cellular physiology since its differentiation.
It seems unnecessary to recall that a similar constancy of sequence, also with no regular pattern of distribution, is found in the triplets of nucleotides that form nucleic acids. However, the significant difference is that the specific function of the nucleic acids does not lie (as in globular proteins) in the entire nucleic acid but in each triplet of nucleotides that, precisely through the mediation of specific globular proteins, leads to the incorporation of a specific L-α-amino acid in the right place of a corresponding polypeptide. In both cases it is unimaginable for linear concatenations of molecules (which are diverse and have equivalent reactivity, with apparently arbitrary but absolutely fixed sequences) to result from merely chemical reactions.
A structural character exclusive to globular proteins is the fact that the polypeptide or its constituent polypeptides are folded so that the residues of L-α-amino acids acquire the globular configuration that makes these proteins functionally active. This folding (studied systematically in the mid-twentieth century using X-ray diffraction diagrams), like the sequence of α-amino acids in polypeptide, is characteristic of each species of globular protein (defined by its function and by the type of cell to which it belongs) and shows no regularity of structural pattern that allows subordinate units (postulated by reductionism to the molecular level and carefully sought) to be discovered. The absence of regular internal patterns of esters in the folding of the polypeptides of the proteins means that (like their sequence of α-amino acids) they must have been produced in the functional refinement of each globular protein during the evolution of its cell. As stated above with respect to the constancy of the configuration of amino acid esters (all L) and the constancy of their sequence in the polypeptides of each type of globular protein, the fact that polypeptides of a given sequence always fold in a single, intricate and rigorously specific way is also inexplicable through the laws of chemistry.
Representation of the metabolic transformations governed by globular proteins
In conclusion, it is incomprehensible that a complex folding of polypeptides characteristic of each type of globular protein is organized only through the interactions of the constituent molecules of polypeptides and of water (i.e. through chemical laws). Either we merely justify it as a mysterious teleological quality of each polypeptide (and give up the possibility of understanding the emergence of the animal from its embryonic cells), or we decide to consider, as a potentially solvable (and very real) problem, how certain polypeptides fold, each one in a perfectly specific way, and form a globular protein with its clearly supramolecular functional capacity, as we will see below. (The statement that the globular protein is a living being -midway between the molecule and the cell- raises the problem of the origin of the first globular protein since the evolution of molecules on the Earth’s surface, long before the origin of nucleic acids at the service of the cell’s proteins). Evolutionist Treatise of Biology). Part One.. (p. 139-223).
(An analysis of the action of metabolic proteins)
Important collections of data of various kinds indicate that globular proteins are living beings, i.e. genuine agents that govern a specific trophic environment to their own benefit by alternately using action and experience. There is no action without experience to guide it, nor experience of anything other than the effects of a performed action.
The environment of the proteins consisted, ab origine and still at present, of molecules dissolved in becalmed water and the action of the proteins in handling them one by one. This action is known experimentally in detail through the analysis of the activity of hundreds of globular proteins with a metabolic function that has been achieved by biochemistry. We have dedicated a large part of our research in the last few years to interpreting this activity in the Tratado evolucionista de biología (Evolutionist Treatise of Biology). Part Two. Volume II.
Using a schema that we devised, we have analysed each of the metabolic transformations discovered by biochemistry, and we have distinguished exactly the action performed by the metabolic protein and the chemical transformation that the protein causes in metabolites and coenzymes. This analysis of each metabolic transformation has enabled us to specify how metabolic proteins govern elementary chemical reactions by applying their particular mode of action and experience.
We will now specify the differences between chemical reactions in free molecules (with or without a catalyst) and the chemical reactions of metabolic transformations; these differences are attributable to the presence of the protein as a supramolecular agent.
As is generally known, in favourable conditions, molecules in gaseous or dissolved state are transformed into others (with exchange of atoms) through the splitting of molecules and through random encounters between pairs of molecules. The frequency of the transformations is related to the concentration of the molecules and to their mobility, which in turn depends on heat. The production of other molecules from the encounter of a pair of molecules depends on their chemical affinity, and on their energy state and reciprocal positions when they come together. We can add that the reaction resulting from the random encounter of two molecules of any complexity, like the majority of organic molecules, depends on whether in this encounter they are suitably polarized so that the two functional groups face each other. For each pair of molecules, this tends to give rise to several possibilities of reaction, from which several types of product are obtained. Each possible reaction is the result of a type of structural disturbance of the two molecules that tends to be recomposed by internal factors, giving other, more stable species of molecule.
Formerly, the attempt to reduce the cellular metabolism to the cellular level led globular proteins to be considered as mere catalysts (though with a function that was not explicitly understood). However, catalysts of all kinds -homogeneous and heterogeneous- obey the laws of chemistry. Indeed, when catalysts operate (dissolved, in gaseous state or forming part of a solid), they behave like genuine molecules that react with the gaseous or liquid states of a reagent through random encounters and lead to statistical results that agree with those corresponding to the equilibrium constant of the uncatalysed reaction. Both functional aspects of the catalysts are inconsistent with the activity of proteins, supramolecular complexes that operate by causing -as will be specified below- certain irreversible chemical reactions in 100% of the encounters in the cell soma. Chomín Cunchillos, Interpretación de la función enzimática de las proteínas a partir de la teoría de unidades de nivel de integración (Spanish)
Metabolites and coenzymes, i.e. molecules that are not free but submitted to the action and experience of a metabolic protein, cease to encounter each other randomly from the moment when, as a result of the transformations in the “active centre” of their specific protein, they are directed by this protein towards the following metabolic protein. In the “active centre” of the same protein, molecules of three, four or more molecular species are led to converge, instant by instant, and they react in a very rapid succession of pairs. This convergence of more than two molecules is statistically very improbable if they operate alone in reactions between free molecules (and it is not taken into account by chemical kinetics), whereas it occurs with total regularity in the metabolic transformations in which the proteins that govern them so require.
In the schemas conceived to represent metabolic transformations, according to our knowledge of “enzymatic mechanisms”, in addition to making the metabolites and coenzymes converge in their “active centre”, the metabolic protein must in all probability keep them still, at interatomic distances and with a precise reciprocal position, and must act on them to cause with absolute regularity a very determined reaction (always precisely formulable in chemical terms), which is almost never reproducible in vitro without the presence of the specific enzyme. Evolutionist Treatise of Biology). Part Two. Volume II
In each metabolic transformation one can plausibly interpret how the action of the protein can operate on the molecular fields of the metabolites and coenzymes and how -in the opposite direction and with alternating tempo- the effect of the action on the molecular fields can affect the experience of the protein that guides its next action. Once the developed formulas of the metabolites and coenzymes that initiate a metabolic transformation are placed in its schema, in the always unequivocal placement required by the result of the transformation, it becomes comprehensible that the action of the metabolic protein, after placing the molecules at the precise distance and position, is limited to polarizing two well-determined points of the established set of molecular fields of the metabolites, which causes a series of jumps of pairs of electrons in the intermediate interatomic bonds of different molecules. This very fine management of a metabolic protein of two or more molecules causes the concerted series of directed jumps of electrons and, as a subsidiary effect, the emission of H+ or OH- to the water of the area or the acceptance of these ions from it. The set of effects of the action of a specific protein (establishment of a bond between two molecules or splitting of a molecule due to the disappearance of a bond, creation, disappearance and displacement of double bonds, hydrogenation or dehydrogenation, introduction of hydroxyls, etc.) causes the following very general results in the single- or multi-cell zone that is affected:
a) It occurs with no irreversible loss of atoms for the cellular metabolism.
b) There is a tenacious tendency to preserve the joint molecular structure of metabolites and coenzymes not only of the part not affected by the metabolic reaction, but even of the portion of metabolites and coenzymes that are affected by the transformation.
c) It is perceptible that the establishment of each metabolic transformation has been the evolutive achievement of a globular protein that offered a selective advantage for the cell and that served as a basis for the differentiation of the next metabolic protein in evolution.
In short, in order for the supramolecular agent, the globular protein with a metabolic function, to perform its complex action guided by its corresponding experience, it seems that the successive moments that occur in it must be: the capture of the metabolites and coenzymes that it receives from its previous metabolic protein; their convergent transport; the coordination of their relative positions at interatomic distances; the simultaneous activation of their molecular fields, which triggers the specific chemical reactions; the perception of the metabolites resulting from the metabolic transformations; and the outgoing transport (donation) to the following metabolic protein. Achieving this is impossible in free molecules, i.e. if it is not driven by a supramolecular agent.
It is understandable that an inevitable consequence of the reduction of the globular protein (of the living being of the directly supramolecular and infracellular level) to the molecular level is the vacuum left by the agent which, nevertheless, has obvious functions in the cell. Paradoxically, it has been attempted to fill this void with the alleged activity of molecules (or associations of molecules), by attributing functions to them that are inconsistent with their capacity as discovered by chemical science.