Part III: https://systemity.livejournal.com/4661963.html
Part I: https://systemity.livejournal.com/4661364.html
Original paper "View on Lipids of Microorganisms from the Standpoint of Prebiotic and Biological Evolution" published in: Voprosy Evolutsii Bakterij (Evolution of Bacteria), USSR Academy of Sciences, Center for Biological Research, Institute of Biochemistry and Physiology of Microorganisms, Pushchino, 1984, pp. 93-119 (https://www.dropbox.com/s/
Enzymes and coenzymes
The membrane system of prokaryotic organisms lacks an expressed anatomic compartmentalization of membrane enzymatic processes, which is the key difference between eukaryotic and prokaryotic organisms. The prokaryotic type of membrane system organization causes tremendous difficulties for the cellular level regulation of the membrane enzymatic processes which involve same coenzymes. Strangely, this peculiarity of bacteria has been getting very little attention . Based on molecular weights of the most widely distributed coenzymes that supply membrane-immobilized enzymes of small reacting molecules, such as protons, C1- and C2- compounds, etc., it can be easily established, by the Einstein diffusion equation, that at physiological temperatures, those enzymes cover within a second the distances that several times exceed the length of a bacterial cell. Knowing that, it is hard to understand, for instance, the secrets of coordination of the activity of dozens of types of enzymes, whose functioning requires pyridine nucleotide carriers in specific concentrations strictly determined by a current physiological state of the cells.
As to eukaryotic organisms, the above problem is solved by the presence of partially autonomous membrane systems which can provide necessary conditions for physiologically adequate functioning of enzymatic systems – firstly, by selective distribution of enzymes in intracellular membrane systems; and, secondly, by creating optimal concentrations of coenzymes and cofactors in the membrane’s closed compartments. Prokaryotes must possess other mechanisms, one of which probably consists in mostly allosteric regulation of the activity of membrane enzymes by changing the composition of lipids that surround the enzymes. In its turn, changes in lipid composition can be done through changing the concentrations of coenzymes involved in lipid biosynthesis. Principle of quasi-equilibrium of biosynthesis of bacterial lipids The concept of polylipids sheds light upon the role of lipids in coordinating the activity of membrane-immobilized enzymes with the activity of coenzymes and cofactors dissolved in the cellular fluid. Changes in individual lipid compositions influence the conformational changes in enzymes and their location in biological membranes, which, in its turn, changes their activity. It seems that for such changes to be in accord with coenzymes three main conditions must be met: (1) there has to be two types of lipid biosynthesis control by coenzymes and cofactors – strict and non-strict; (2) the control has to be provided in the near-equilibrium area; and (3) allosteric regulation of enzymes of a given particular organism or group of organisms must be specifically adapted to the effect of the lipid environment.
The first of the three conditions is required because, along with the problem of regulation of individual enzymes, there is a problem of coordination of the activities of enzymes in enzymatic systems. In enzymatic systems, the roles of same coenzymes can be drastically different. The third condition is determined by the well-known fact that the mechanisms for allosteric regulation of the membrane’s homologous enzymes, isolated from different organisms, are different and use different spectra of isoenzymes. We will focus more closely on the second condition as its practical realization in bacteria can be easily proven.
All eubacteria synthesize saturated normal fatty acids. Their biosynthesis occurs due to cooperation of seven enzymatic systems: acetyl-CoA carboxylase, acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, β-ketoacyl synthetase, β-ketoacyl reductase, β-hydroxyacyl dehydrase, and enoyl reductase . The last four stages occur upon each successive bonding of the C2 unit. All partial reactions are reversible – however, it does not mean that the anabolic and catabolic processes involved in regulation of biosynthesis of normal saturated fatty acids differ only in the reaction direction. Differences between direct and reverse reactions become more expressed in case of fatty acids with C1-modified fatty acid residues, such as cyclopropanic, monomethyl-substituted. In view of the above, the equilibrity of biosynthesis of fatty acids in bacteria exists not due to but in spite of the known molecular mechanisms. Nonetheless, and irrespective of whether or not we are ready to understand the general and fine mechanisms of this phenomenon, equilibrity is the fundamental principle of biosynthesis of lipids – both whole molecules and parts of molecules, i.e. fatty acid residues – in bacteria. Clearly, the means for maintaining such equilibrium are very much different from those used in non- biological systems, and therefore the term ‘quasi-equilibrium’ is more appropriate for this phenomenon.
Indeed, in bacterial populations, there are certain barriers that prevent the establishing of equilibrium by regular means known in thermodynamics of chemical processes: intercellular, intermembrane, axial, and intramembrane lateral barriers, of which only the latter seems to be more or less surmountable. As well, it is hard to explain how the equilibrium constants stay unchanged in a bacterium population where the ratio between live and dead cells varies according to certain laws that are both complex and unknown to us.
The (quasi-) equilibrity of lipid biosynthesis can be easily discovered based on the law of mass action. Under (quasi-) equilibrium, the ratio of the mathematical product of concentrations of synthesis products to the mathematical product of initial substrates’ concentrations raised to the power that equals their stoichiometric coefficients, at a given constant temperature, must remain constant at changing concentrations of individual substances. Examples of equilibrity of biosynthesis of phospholipids and fatty acids in various species of bacteria were previously provided by us in [2, 4-7, 19]. In the context of this paper, it should be important to discuss one of the examples demonstrating that the effect of environmental physical factors on the lipid composition of bacterial membranes can be explained and predicted without the recourse to biophysics-based simplifications such as “melting and solidification of membrane lipids”.
Fig. 2 shows a correlative dependence between elongation indexes of fatty acids of four strains of bacteria of the genera Arthrobacter, Curtobacterium and Microbacterium, grown for 63 hours on slanted meat-peptone agar medium at 19o and 29o C. According to the principle of (quasi-) equilibrity of lipid biosynthesis, the elongation index A of a series m fatty acid with the n number of C atoms equals:
where a and a are concentrations of the coenzyme involved in the transfer of a C2 unit to fatty acid mn in free (CoA) and activated (malonyl-CoA, acetyl-CoA) forms, respectively; and mn K is the constant of quasi-equilibrity of the reaction of elongation of fatty acid mn; concentrations of the substances are indicated in square brackets. The plot shown in Fig. 2 demonstrates that the quasi-equilibrity principle is strictly observed in the acids that not only have different melting point temperatures but also their melting point temperature dependences are different. It appears that the pattern of the fatty acid chain elongation in the course of raising the cultivation temperature by 10o
does not depend on the concentration of each acid at the initial temperature of 19o C. For instance, 19o C the content of fatty acids a15:0, a17:0, i15:0 and i17:0 in Arthrobacter oxydans VKM 663 was 72.57, 9.95, 3.63, and 0.14%, respectively; whereas at 29o C the amounts of the same fatty acids were: 63.71, 16.43, 4.58 and 0.45%, respectively. The assessment of temperature-based fluctuations of individual fatty acids’ presence in a bacterium population does not reveal any specific pattern, and at first glance those fluctuations seem to be chaotic. The linearity of the curve shown in Fig. 2 is a result of (a) quasi-equilibrity and (b) identical temperature-dependences for the quasi-equilibrity constants and coenzyme concentrations, which can be explained by the physiological and biochemical affinity between the coryneform bacteria strains under study.
The quasi-equilibrity principle helps to understand the origin of the diversity of fatty acid spectra in eubacteria and its relationship with environmental factors. First of all, within the limits of the four groups of the afore-mentioned bacteria, it sheds light upon certain general regularities that are common for the genera and families represented by each of the four groups. For instance, in Gram-negative bacteria, Mycobacteria and alike, as well as Lactobacilli, the main reactions occurring during the quasi-equlibrium modification of fatty acids are dehydration, methylation, and elongation; and in all cases, without any exceptions, growth and development consist of two stages. During the first stage of bacterial growth and development, lipids in general and fatty acids in particular undergo demethylation; whereas the second stage is accompanied with methylation. There have been found no exceptions from this rule. Lipids act as a depot of freely convertible methyls that are released at the peak of a bacterium’s physiological activity. In Gram-positive bacteria that synthesize branched fatty acids (group 2), no lipid methylation occurs at all. Dehydration occurs in only some of the bacteria of this group and most often is a species or strain feature. In such bacteria, the fatty acid composition is regulated by their predecessors – mainly, amino acids – which, upon their deamination into ketoacids, initiate the biosynthesis of various branched acids [26, 27].
Thus, the quasi-equilibrity principle*** provides the understanding of physiological and biochemical significance of the diversity and specificity of lipid composition of bacteria. Obviously, one can speak of the morphological role of the lipid composition, as in many bacteria morphological differentiation seems to be designed to provide compartmentalization of membrane enzymatic processes as a solution for resolving the conflicts between enzymes and coenzymes. As was mentioned above, groups of bacteria which have distinctive peculiarities of lipid metabolism, have also distinctive specifics of their cytostructural organization, general metabolism, physiology and ecology. No doubt that these factors – as complex as all the phenomena of the biological level of complexity – had been the determining factors in natural selection and evolution of the ways for lipid metabolism organization. Therefore, no matter how thoroughly and scrupulously we explore the lipid metabolism of live organisms, the questions of why it is organized in a certain particular way and not in any other, which of its features are common in all organisms, and why it is different in different groups of organisms – will remain unanswered as long as we do not have a sufficiently clear understanding of how the life had emerged and evolved on Earth.
*** For more information about theoretical and practical applications of the quasi-equilibrity principle, see, e.g.:
L. Andreev. Quasi-equilibrium as a general principle of regulation of lipid composition of eubacteria. In: Environmental regulation of microbial metabolism. I.S. Kulaev, E. A. Dawes, D. W. Tempest (Eds.). Academic Press, London, pp. 161-185, 1985.
L. Andreev. Taxonomic calculations based on fatty acid spectra of bacteria. Requirements for chromatographic analysis of fatty acids. In: Rapid Methods and Automation in Microbiology and Immunology. K.-O. Habermehl (Ed.), Springer Verlag, Berlin, Heidelberg, New York, pp. 265-273, 1985.
L. Andreev et al. Grouping of Staphylococcus species based on their fatty acid spectra. In: The Staphylococci. J. Jeljaszewicz (Ed.). Gustav Fisher Verlag, Stuttgart, New York, pp. 151-155, 1985.
On the origin of life on Earth
In an article on the origin of life on Earth, Woese – criticizing Oparin’s prebiotic broth hypothesis – theorizes that life could have emerged in microdroplets of water that was present, in the aerosolized state, in the prebiotic atmosphere containing hydrogen and carbonic acid . Per Woese, microdroplet water suspension was that very biochemical reactor in which the basic metabolism could have emerged, which, in its turn, should have resulted in the emergence of methanogens, the first possible life form according to Woese. This original theory of Woese’s shares at least one of the points of Oparin’s theory [13, 15]: that life must have first emerged in a phase-separated system, i.e. the medium in which the main events were developing was characterized by a higher ratio of phase interface to phase volumes. Phase interface have higher levels of free surface energy, hence an increased capability to concentrate substances from the volumes of adjacent phases and thus facilitate the occurrence of chemical reactions. Phase-separated systems have a number of peculiarities that are significant in the context of scientific reconstruction of the prebiotic evolution processes .
The examination of those peculiarities can help to narrow the action arena of the forces that have produced Life and to propose a certain general scenario whose implementation in any of the countless possible ways could have led to the emergence of live organisms. However, by the modern science standards, this kind of substantiation is not sufficient for asserting that life on Earth has spontaneously self-emerged on Earth and not as a result of panspermia or the six-day creation by God. The majority of contemporary scientists tend to maintain that the emergence of life was a pre-determined result of the substance and energy properties exhibited in the course of progressive evolution of the more and more complex carbon compounds and macromolecular open systems formed from them . This idea has inspired much optimism and created many believers in the feasibility of discovery of the origin of life by exploring into molecular foundations of the functioning of current forms of life. However, the concept of spontaneous self-emergence of life on Earth remains to be more of an article of faith than a subject of strong scientific proof. To many scientists, the life origin scenarios which explain both the most important and the least probable processes by referring to predetermination, spontaneity, and various tunnel effects are no longer credible, and some of the researchers turned to believe that terrestrial life could not get started on Earth****.
In live organisms, various substances – from metal ions to huge macromolecules – are involved in an incredibly complex network of interrelations, in which the subordination is both strict and flexible. The cause of such duality may lie in the conflict between the historic order of formation of the substances that have a general biological importance and their functional roles in the currently existing live organisms. Even though the probability of spontaneous emergence of macromolecules with a biologically purposeful structure is estimated as a decimal fraction with hundreds of zeros at the right of the numerator, most scientists agree that it was the formation of functionally active biopolymers that gave the impetus to the emergence of life, whereas a major point of disputes is: which of the two – proteins or nucleic acids – were formed first?
Pre-biological self-development of macromolecules is viewed upon as certain harmony between the physico-chemical nature of substances and the environment, which eventually leads to self-assembly of a functionally holistic structure out of parts that have emerged and reached structural and functional perfection outside that structure. If the emergence of life was thrust by the organization of a system of such macromolecules as DNA, RNA, or a protein, which had previously become “almost live” due to purely physico-chemical mechanisms, then it should be perfectly logical to look for such mechanisms which, of course, must be capable of spontaneous self-implementation.
There is a great deal of publications describing possible scenarios of the emergence of life. Some of the scenarios are based on certain pre-defined initial conditions – cf. hypotheses by Oparin [13-15], Fox , Woese . Others focus on certain general laws of physics and chemistry which could provide the foundation for self-development of functionally active biopolymers and their self-assembly into something similar to a live cell – e.g. the principle of kinetic perfection proposed by Shnol , a hypercycle according to Eigen [23, 24], etc. Following those discussions, the notion of natural selection of macromolecules has become popular but has not provided any clarity as to a definite purpose of such selection. The purpose is being set by the proposing researchers based on retrospective analysis of the known properties of the living matter. Here is, for instance, a plan for animation of inorganic substances, proposed by Eigen and Schuster in their trilogy, Hypercycle [23, 24]. The whole process consists of six stages (the quotes below are from a discussion by Volkenstein ):
“1. The emergence of macromolecules is determined by their structural stability and monomer contents. The early stage polymers were protein-like chains and a few RNA-like polymers capable of replication.
2. The composition of primary polynucleotides was determined by concentrations of monomers. Sequence reproduction depends on the accuracy of copying, which is higher in GC sequences. Reproduced sequences formed the quasi-species distribution.”
After the next three points, there follows:
“6. That organization further developed by taking advantage of favorable phenotypic changes. It appeared that a fair selection of
specific genotypes required spatial separation”.
The above plan is a good example of transformation of logical cognition techniques through gradual replacement of induction by deduction, which is getting increasingly widespread in the contemporary science about the origin of life. As of now, there is a lot of established truths that raise no doubt – such as, for instance, the role of DNA as a carrier of genetic information – which leads many scientists to believe that the deduction of such truths from other, more general, truths is more productive than the formulation of scientific laws based on concrete experimental data and models.
The said tendency is clearly seen in theoretical studies into the origin of life. However realistic the verification of the heretofore proposed hypotheses may seem to be, as of today they are as dogmatic as creationism, as, in the essence, they boil down to the postulate about spontaneous self-formation of biologically purposeful macromolecules. In reality, a discussion – at whatever high scientific level – of the process of self-formation of such macromolecules can be scientific only in the part concerning the post factum, not prior, events. Therefore, scenarios like Hypercycle are regarded by serious researchers in the origin of life as “The Game of Life”, even if it is played by talented physicists, chemists, and mathematicians.
Volkenstein notes that the model by Eigen and Winkler obviously does not claim to be a re-enactment of actual events that had been occurring on Earth at the time when life was emerging but aims at proving the possibility of self-organization of the matter, based on the known principles of physics . So, it would appear that the main road leading down has been cleared, and all what is left to be done is to solve particular problems – for instance, to find the right place for DNA, the “soul” of the biological cell, so it should be able to continue the process of spontaneous development. But this, too, appears to have been theoretically solved, and the path from the general to particulars seems to be even cleaner – this, of course, refers to phase-separated systems, such as, for instance, coacervate droplets as proposed by A. Oparin. In one of his latest works, he wrote: “Initially, at the molecular level, only protein- and nuclein-like polymers, devoid of any “biological purposefulness” in their intramolecular structure, could emerge... Only when combined in multi-molecular phase-separated systems, those polymers, interacting with each other, were capable of mutual coordination of their intramolecular structures and functions within a system. Natural selection of phase-separated systems had also determined the emergence of biological “purposefulness” and specificity of polymer structures, as well as the life-specific form of information transfer (heredity) .
**** The original Russian text includes a quote from an article by Ch. Wickramasinghe in UNESCO Courier .
In fact, all of the known hypotheses about the origin of life differ from each other only in the part concerning the view on the kind of setting that is most likely to have been proper for a sudden materialization of the key “figure” – DNA. The main requirement to such a setting is that it should represent a structure of the live cell type but made of inorganic matter. Phase-separate systems certainly seem to be by far more natural as an environment for the emergence of life, than, for instance, simple water solutions. Nevertheless, natural selection, by itself, of phase-separated systems can result in neither the “biological purposefulness” and specificity of polymer structures, nor the emergence of the “life-specific form of information transfer (heredity)”. That would be possible only if phase-separated systems themselves had a biologically purposeful structure or were capable of performing biological functions at least at the primordial, maximally reduced level. To assume that the first of these two conditions was met would equal the assumption that a biologically purposeful structure of the primitive phase-separated systems had spontaneously emerged by itself – which is even less probable. As to the functionality, it could have developed only in the presence of an adequate structure.