The Mystery of Life's Origin:

Reassessing Current Theories



CHAPTER 9

Specifying How Work Is To Be Done

In Chapter 7 we saw that the work necessary to polymerize DNA and protein molecules from simple biomonomers could potentially be accomplished by energy flow through the system. Still, we know that such energy flow is a necessary but not sufficient condition for polymerization of the macromolecules of life. Arranging a pile of bricks into the configuration of a house requires work. One would hardly expect to accomplish this work with dynamite, however. Not only must energy flow through the system, it must be coupled in some specific way to the work to be done. This being so, we devoted Chapter 8 to identifying various components of work in typical polymerization reactions. In reviewing those individual work components, one thing became clear. The coupling of energy flow to the specific work requirements in the formation of DNA and protein is particularly important since the required configurational entropy work of coding is substantial.

Theoretical Models for the Origin of DNA and Protein

A mere appeal to open system thermodynamics does little good. What must be done is to advance a workable theoretical model of how the available energy can be coupled to do the required work. In this chapter various theoretical models for the origin of DNA and protein will be evaluated. Specifically, we will discuss how each proposes to couple the available energy to the required work, particularly the configurational entropy work of coding.

Chance

Before the specified complexity of living systems began to be appreciated, it was thought that, given enough time, "chance" would explain the origin of living systems. In fact, most textbooks state that chance is the basic explanation for the origin of life. For example, Lehninger in his classic textbook Biochemistry states,
We now come to the critical moment in evolution in which the first semblance of "life" appeared, through the chance association of a number of abiotically formed macromolecular components, to yield a unique system of greatly enhanced survival value.1
More recently the viability of "chance" as a mechanism for the origin of life has been severely challenged.2

We are now ready to analyze the "chance" origin of life using the approach developed in the last chapter. This view usually assumes that energy flow through the system is capable of doing the chemical and the thermal entropy work, while the configurational entropy work of both selecting and coding is the fortuitous product of chance.

To illustrate, assume that we are trying to synthesize a protein containing 101 amino acids. In eq. 8-14 we estimated that the total free energy increase (G) or work required to make a random polypeptide from previously selected amino acids was 300 kcal/mole. An additional 159 kcal/mole is needed to code the polypeptide into a protein. Since the "chance" model assumes no coupling between energy flow and sequencing, the fraction of the polypeptide that has the correct sequence may be calculated (eq. 8-16) using equilibrium thermodynamics, i.e.,

[protein concentration] / [polypeptide concentration] = exp ( - G / RT), eq. (9-1)

= exp (-159,000) / 1.9872 x 298)

or approximately 1 x 10-117

This ratio gives the fraction of polypeptides that have the right sequence to be a protein.

[NOTE: This is essentially the inverse of the estimate for the number of ways one can arrange 101 amino acids in a sequence (i.e., I / c in eq. 8-7)].
Eigen3 has estimated the number of polypeptides of molecular weight 10 4 (the same weight used in our earlier calculations) that would be found in a layer 1 meter thick covering the surface of the entire earth. He found it to be 1041. If these polypeptides reformed with new sequences at the maximum rate at which chemical reactions may occur, namely 1014/s, for 5 x 109 years [1.6 x 1017 s], the total number of polypeptides that would be formed during the assumed history of the earth would be

1041 x 1014/s x 1.6 x 1017s = 1072 (9-2)

Combining the results of eq. 9-1 and 9-2, we find the probability of producing one protein of 101 amino acids in five billion years is only 1/ 1045. Using somewhat different illustrations, Steinman4 and Cairns-Smith5 also come to the conclusion that chance is insufficient.

It is apparent that "chance" should be abandoned as an acceptable model for coding of the macromolecules essential in living systems. In fact, it has been, except in introductory texts and popularizations.

Neo-Darwinian Natural Selection

The widespread recognition of the severe improbability that self-replicating organisms could have formed from purely random interactions has led to a great deal of speculation---speculation that some organizing principle must have been involved. In the company of many others, Crick6 has considered that the neo-Darwinian mechanism of natural selection might provide the answer. An entity capable of self-replication is necessary, however, before natural selection can operate. Only then could changes result via mutations and environmental pressures which might in turn bring about the dominance of entities with the greatest probabilities of survival and reproduction.

The weakest point in this explanation of life's origin is the great complexity of the initial entity which must form, apparently by random fluctuations, before natural selection can take over. In essence this theory postulates the chance formation of the "metabolic motor" which will subsequently be capable of channeling energy flow through the system. Thus harnessed by coupling through the metabolic motor, the energy flow is imagined to supply not only chemical and thermal entropy work, but also the configurational entropy work of selecting the appropriate chemicals and then coding the resultant polymer into an aperiodic, specified, biofunctioning polymer. As a minimum, this system must carry in its structure the information for its own synthesis, and control the machinery which will fabricate any desired copy. It is widely agreed that such a system requires both protein and nucleic acid.7 This view is not unanimous, however. A few have suggested that a short peptide would be sufficient.8

One way out of the problem would be to extend the concept of natural selection to the pre-living world of molecules. A number of authors have entertained this possibility, although no reasonable explanation has made the suggestion plausible. Natural selection is a recognized principle of differential reproduction which presupposes the existence of at least two distinct types of self-replicating molecules. Dobzhansky appealed to those doing origin-of-life research not to tamper with the definition of natural selection when he said:

I would like to plead with you, simply, please realize you cannot use the words "natural selection" loosely. Prebiological natural selection is a contradiction in terms.9
Bertalanffy made the point even more cogently:
Selection, i.e., favored survival of "better" precursors of life, already presupposes self-maintaining, complex, open systems which may compete; therefore selection cannot account for the origin of such systems.10
Inherent Self-Ordering Tendencies in Matter

How could energy flow through the system be sufficiently coupled to do the chemical and thermal entropy work to form a nontrivial yield of polypeptides (as previously assumed in the "chance" model)? One answer has been the suggestion that configurational entropy work, especially the coding work, could occur as a consequence of the self-ordering tendencies in matter. The experimental work of Steinman and Cole11 in the late Sixties is still widely cited in support of this model.12 The polymerization of protein is hypothesized to be a nonrandom process, the coding of the protein resulting from differences in the chemical bonding forces. For example, if amino acids A and B react chemically with one another more readily than with amino acids C, D, and E, we should expect to see a greater frequency of AB peptide bonds in protein than AC, AD, AE, or BC, BD, BE bonds.

Together with our colleague Randall Kok, we have recently analyzed the ten proteins originally analyzed by Steinman and Cole,13 as well as fifteen additional proteins whose structures (except for hemoglobin) have been determined since their work was first published in 1967. Our expectation in this study was that one would only get agreement between the dipeptide bond frequencies from Steinman and Cole's work and those observed in actual proteins if one considered a large number of proteins averaged together. The distinctive structures of individual proteins would cause them to vary greatly from Steinman and Cole's data, so only when these distinctives are averaged out could one expect to approach Steinman and Cole's dipeptide bond frequency results. The reduced data presented in table 9-1 shows that Steinman and Cole's dipeptide bond frequencies do not correlate well with the observed peptide bond frequencies for one, ten, or twenty-five proteins. It is a simple matter to make such calculations on an electronic digital computer. We surmise that additional assumptions not stated in their paper were used to achieve the better agreements.

Furthermore, the peptide bond frequencies for the twenty-five proteins approach a distribution predicted by random statistics rather than the dipeptide bond frequency measured by Steinman and Cole. This observation means that bonding preferences between various amino acids play no significant role in coding protein. Finally, if chemical bonding forces were influential in amino acid sequencing, one would expect to get a single sequence (as in ice crystals) or no more than a few sequences, instead of the large variety we observe in living systems. Yockey, with a different analysis, comes to essentially the same conclusion.14

A similar conclusion may be drawn for DNA synthesis. No one to date has published data indicating that bonding preferences could have had any role in coding the DNA molecules. Chemical bonding forces apparently have minimal effect on the sequence of nucleotides in a polynucleotide.

Table 9-1.

Comparison of Steinman and Cole's experimentally determined dipeptide bond frequencies, and frequencies calculated by Steinman and Cole, and by Kok and Bradley from known protein sequences.



Dipeptide*

Values (relative to Gly-Gly)


S / C+

K / B #


exp &

cal

cal-wa

cal-woa


Gly-Gly

1.0

1.0

1.0 (1.0) [1.0]



1.0 (1.0) [1.0]


Gly-Ala



0.8

0.7

1.1 (1.1) [2.0]



2.0 (1.2) [1.0]


Ala-Gly



0.8

0.6

1.0 (1.1) [2.2]



1.5 (1.2) [0.0]


Ala-Ala



0.7

0.6

1.3 (1.5) [4.4]



2.8 (1.5) [0.0]


Gly-Val



0.5

0.2

0.2 (0.3) [0.4]



1.5 (1.2) [1.0]


Val-Gly



0.5

0.3

0.3 (0.3) [0.6]



0.8 (0.6) [0.0]


Gly-Leu



0.5

0.3

0.3 (0.3) [0.2]



1.3 (0.7) [1.0]


Leu-Gly



0.5

0.2

0.3 (0.3) [0.8]



1.3 (1.0) [1.0]


Gly-Ile



0.3

0.1

0.1 (0.2) [0.6]



1.0 (0.8) [0.0]


Ile-Gly



0.3

0.1

0.1 (0.2) [0.2]



0.0 (0.4) [0.0]


Gly-Phe

0.1

0.1

0.1 (0.2) [0.4]

0.5 (0.5) [0.0]


Phe-Gly

0.1

0.1

0.1 (0.1) [0.6]

1.0 (0.5) [1.0]

(Adapted after G. Steinman and M.V. Cole, 1967. Proc. Nat. Acad. Sci. U.S. 58,735).

* The dipeptides are listed in terms of increasing volume of the side chains of the constituent residues. Gly = glycine, Ala = alanine, Val = valine, Leu = leucine, Ile = isoleucine and Phe = phenylalanine. Example: Gly-Ala = glycylalanine.

+ Steinman and Cole's (S/C) experimentally determined dipeptide bond frequencies were normalized and compared to the calculated frequencies obtained by counting actual peptide bond frequencies in ten proteins, assuming all seryl and threonyl residues are counted as glycine and all aspartyl and glutamyl residues are counted as alanine. The ten proteins used were: egg lysozyme, ribonuclease, sheep insulin, whale myoglobin, yeast cytochrome c, tobacco mosaic virus, beta-corticotropin, glucagon, melanocyte-stimulating hormone, and chymotrypsinogen. Because of ambiguity regarding sequences used by S/C, all sequences are those shown in Atlas of Protein Sequence and Structure, 1972. Vol. V (ed. by M.O. Dayhoff). National Biomedical Research Foundation, Georgetown University Medical Center, Washington, D.C.

& The experimentally determined dipeptide frequencies were obtained with aqueous solutions containing 0.01 M each amino acid, 0.125 N HCl, 0.1 M sodium dicyanamide.

#Kok and Bradley's (K/Bcalculated dipeptide frequencies were obtained by counting S?Cassumptions. The numbers in brackets are for one protein, enterotoxin B, with actual peptide bond frequencies for the same ten proteins with (wa) and without (woa) S/C assumptions. The numbers in parentheses are for twenty-five proteins with (wa) and without (woa) S/C assumptions. The twenty-five proteins are the ten used S/C and alpha S1 Casein (bovine); azurin (bordetella bronchisetica); carboxypeptidase A (bovine); cytochrome b5 (bovine); enterotoxin B; elastase (pig); glyceraldehyde 3-phosphate dehydrogenase (lobster); human growth hormone; human hemoglobin beta chain; histone 11B2 (bovine); immunoglobulin gamma-chain 1, V-I (human EU); penicillinase (bacillus licheniformis 749/c); sheep prolactin; subtilisin (bacillus amyloliquefaciens); and tryptophan synthetase alpha chain (E-coh K-i 2). Sequences are those shown in Atlas of Protein Sequence and Structure, 1972. Vol. V (ed. by M.O. Dayhoff). Note disagreement S/C K/B calculated results. Also S/C calculated results are at variance with S/C experimental values for one, ten or twenty-five proteins, with (wa) or without (woa) S/C assumptions.

Mineral Catalysis

Mineral catalysis is often suggested as being significant in prebiotic evolution. In the experimental investigations reported in the early 1970's15 mineral catalysis in polymerization reactions was found to operate by adsorption of biomonomers on the surface or between layers of clay. Monomers were effectively concentrated and protected from rehydration so that condensation polymerization could occur. There does not appear to be any additional effect. In considering this catalytic effect of clay, Hulett has advised, "It must be remembered that the surface cannot change the free energy relationships between reactants and products, but only the speed with which equilibrium is reached."16

Is mineral catalysis capable of doing the chemical work and/or thermal entropy work? The answer is a qualified no. While it should assist in doing the thermal entropy work, it is incapable of doing the chemical work since clays do not supply energy. This is why successful mineral catalysis experiments invariably use energy-rich precursors such as aminoacyl adenylates rather than amino acids.17

Is there a real prospect that mineral catalysis may somehow accomplish the configurational entropy work, particularly the coding of polypeptides or polynucleotides? Here the answer is clearly no. In all experimental work to date, only random polymers have been condensed from solutions of selected ingredients. Furthermore, there is no theoretical basis for the notion that mineral catalysis could impart any significant degree of information content to polypeptides or polynucleotides. As has been noted by Wilder-Smith,18 there is really no reason to expect the low-grade order resident on minerals to impart any high degree of coding to polymers that condense while adsorbed on the mineral's surface. To put it another way, one cannot get a complex, aperiodic-sequenced polymer using a very periodic (or crystalline) template.

In summary, mineral catalysis must be rejected as a mechanism for doing either the chemical or configurational entropy work required to polymerize the macromolecules of life. It can only assist in polymerizing short, random chains of polymers from selected high-energy biomonomers by assisting in doing the thermal entropy work.

Nonlinear, Nonequilibrium Processes

1. Ilya Prigogine

Prigogine has developed a more general formulation of the laws of thermodynamics which includes nonlinear, irreversible processes such as autocatalytic activity. In his book Self Organization in Nonequilibrium Systems (1977)19 co-authored with Nicolis, he summarized this work and its application to the organization and maintenance of highly complex structures in living things. The basic thesis in the book is that there are some systems which obey non-linear laws---laws that produce two distinct kinds of behavior. In the neighborhood of thermodynamic equilibrium, destruction of order prevails (entropy achieves a maximum value consistent with the system constraints). If these same systems are driven sufficiently far from equilibrium, however, ordering may appear spontaneously.

Heat flow by convection is an example of this type of behavior. Heat conduction in gases normally occurs by the random collision of gas molecules. Under certain conditions, however, heat conduction may occur by a heat-convection current---the coordinated movement of many gas molecules. In a similar way, water flow out of a bathtub may occur by random movement of the water molecules under the influence of gravity. Under certain conditions, however, this random movement of water down the drain is replaced by the familiar soapy swirl---the highly coordinated flow of the vortex. In each case random movements of molecules in a fluid are spontaneously replaced by a highly ordered behavior. Prigogine et al.,20 Eigen,21 and others have suggested that a similar sort of self-organization may be intrinsic in organic chemistry and can potentially account for the highly complex macromolecules essential for living systems.

But such analogies have scant relevance to the origin-of-life question. A major reason is that they fail to distinguish between order and complexity. The highly ordered movement of energy through a system as in convection or vortices suffers from the same shortcoming as the analogies to the static, periodic order of crystals. Regularity or order cannot serve to store the large amount of information required by living systems. A highly irregular, but specified, structure is required rather than an ordered structure. This is a serious flaw in the analogy offered. There is no apparent connection between the kind of spontaneous ordering that occurs from energy flow through such systems and the work required to build aperiodic information-intensive macromolecules like DNA and protein. Prigogine, et al.22 suggest that the energy flow through the system decreases the system entropy, leading potentially to the highly organized structure of DNA and protein. Yet they offer no suggestion as to how the decrease in thermal entropy from energy flow through the system could be coupled to do the configurational entropy work required.

A second reason for skepticism about the relevance of the models developed by Prigogine, et al.23 and others is that ordering produced within the system arises through constraints imposed in an implicit way at the system boundary. Thus, the system order, and more importantly the system complexity, cannot exceed that of the environment.

Walton24 illustrates this concept in the following way. A container of gas placed in contact with a heat source on one side and a heat sink on the opposite side is an open system. The flow of energy through the system from the heat source to the heat sink forms a concentration relative to the gas in the cooler region. The order in this system is established by the structure: source-intermediate systems-sink. If this structure is removed, allowing the heat source to come into contact with the heat sink, the system decays back to equilibrium. We should note that the information induced in an open system doesn't exceed the amount of information built into the structural environment, which is its source.

Condensation of nucleotides to give polynucleotides or nucleic acids can be brought about with the appropriate apparatus (i.e., structure) and supplies of energy and matter. Just as in Walton's illustration, however, Mora25has shown that the amount of order (not to mention specified complexity) in the final product is no greater than the amount of information introduced in the physical structure of the experiment or chemical structure of the reactants. Non-equilibrium thermodynamics does not account for this structure, but assumes it and then shows the kind of organization which it produces. The origin and maintenance of the structure are not explained, and as Harrison26 correctly notes this question leads back to the origin of structure in the universe. Science offers us no satisfactory answer to this problem at present.

Nicolis and Prigogine27 offer their trimolecular model as an example of a chemical system with the required nonlinearity to produce self ordering. They are able to demonstrate mathematically that within a system that was initially homogeneous, one may subsequently have a periodic, spatial variation of concentration. To achieve this low degree of ordering, however, they must require boundary conditions that could only be met at cell walls (i.e., at membranes), relative reaction rates that are atypical of those observed in condensation reactions, a rapid removal of reaction flow products, and a trimolecular reaction (the highly unlikely simultaneous collision of three atoms). Furthermore the trimolecular model requires chemical reactions that are essentially irreversible. But condensation reactions for polypeptides or polynucleotides are highly reversible unless all water is removed from the system.

They speculate that the low degree of spatial ordering achieved in the simple trimolecular model could potentially be orders of magnitude greater for the more complex reactions one might observe leading up to a fully replicating cell. The list of boundary constraints, relative reaction rates, etc. would, however, also be orders of magnitude larger. As a matter of fact, one is left with so constraining the system at the boundaries that ordering is inevitable from the structuring of the environment by the chemist. The fortuitous satisfaction of all of these boundary constraints simultaneously would be a its miracle in its own right.

It is possible at present to synthesize a few proteins such as insulin in the laboratory. The chemist supplies not only energy to do the chemical and thermal entropy work, however, but also the necessary chemical manipulations to accomplish the configurational entropy work. Without this, the selection of the proper composition and the coding for the right sequence of amino acids would not occur. The success of the experiment is fundamentally dependent on the chemist.

Finally, Nicolis and Prigogine have postulated that a system of chemical reactions which explicitly shows autocatalytic activity may ultimately be able to circumvent the problems now associated with synthesis of prebiotic DNA and protein. It remains to be demonstrated experimentally, however, that these models have any real correspondence to prebiotic condensation reactions. At best, these models predict higher yields without any mechanism to control sequencing. Accordingly, no experimental evidence has been reported to show how such models could have produced any significant degree of coding. No, the models of Prigogine et al., based on non-equilibrium thermodynamics, do not at present offer an explanation as to how the configurational entropy work is accomplished under prebiotic conditions. The problem of how to couple energy flow through the system to do the required configurational entropy work remains.

2. Manfred Eigen

In his comprehensive application of nonequilibrium thermodynamics to the evolution of biological systems, Eigen28 has shown that selection could produce no evolutionary development in an open system unless the system were maintained far from equilibrium. The reaction must be autocatalytic but capable of self-replication. He develops an argument to show that in order to produce a truly self-replicating system the complementary base-pairing instruction potential of nucleic acids must be combined with the catalytic coupling function of proteins. Kaplan29 has suggested a minimum of 20-40 functional proteins of 70-100 amino acids each, and a similar number of nucleic acids would be required by such a system. Yet as has previously been noted, the chance origin of even one protein of 100 amino acids is essentially zero.

The shortcoming of this model is the same as for those previously discussed; namely, no way is presented to couple the energy flow through the system to achieve the configurational entropy work required to create a system capable of replicating itself.

Periodically we see reversions (perhaps inadvertent ones) to chance in the theoretical models advanced to solve the problem. Eigen's model illustrates this well. The model he sets forth must necessarily arise from chance events and is nearly as incredible as the chance origin of life itself. The fact that generally chance has to be invoked many times in the abiotic sequence has been called by Brooks and Shaw "a major weakness in the whole chemical evolutionary theory."30

Experimental Results in Synthesis of Protein and DNA

Thus far we have reviewed the various theoretical models proposed to explain how energy flow through a system might accomplish the work of synthesizing protein and DNA macromolecules, but found them wanting. Nevertheless, it is conceivable that experimental Support for a spontaneous origin of life can be found in advance of the theoretical explanation for how this occurs. What then can be said of the experimental efforts to synthesize protein and DNA macromolecules? Experimental efforts to this end have been enthusiastically pursued for the past thirty years. In this section, we will review efforts toward the prebiotic syntheses of both protein and DNA, considering the three forms of energy flow most commonly thought to have been available on the early earth. These are thermal energy (volcanoes), radiant energy (sun), and chemical energy in the form of either condensing agents or energy-rich precursors. (Electrical energy is excluded at this stage of evolution as being too "violent," destroying rather than joining the biomonomers.)

Thermal Synthesis

Sidney Fox31 has pioneered the thermal synthesis of polypeptides, naming the products of his synthesis proteinoids. Beginning with either an aqueous solution of amino acids or dry ones, he heats his material at 2000oC for 6-7 hours.
[NOTE: Fox has modified this picture in recent years by developing "low temperature" syntheses, i.e., 90-120oC. See S. Fox, 1976. J Mol Evol 8, 301; and D. Rohlfing, 1976. Science 193, 68].
All initial solvent water, plus water produced during Polymerization, is effectively eliminated through vaporization. This elimination of the water makes possible a small but significant yield of polypeptides, some with as many as 200 amino acid units. Heat is introduced into the system by conduction and convection and leaves in the form of steam. The reason for the success of the polypeptide formation is readily seen by examining again equations 8-15 and 8-16. Note that increasing the temperature would increase the product yield through increasing the value of exp (- G / RT. But more importantly, eliminating the water makes the reaction irreversible, giving an enormous increase in yield over that observed under equilibrium conditions by the application of the law of mass action.

Thermal syntheses of polypeptides fail, however, for at least four reasons. First, studies using nuclear magnetic resonance (NMR) have shown that thermal proteinoids "have scarce resemblance to natural peptidic material because beta, gamma, and epsilon peptide bonds largely predominate over alpha-peptide bonds."32
[NOTE: This quotation refers to peptide links involving the beta-carboxyl group of aspartic acid, the gamma-carboxyl group of glutamic acid, and the epsilon-amino group of lysine which are never found in natural proteins. Natural proteins use alpha-peptide bonds exclusively].
Second, thermal proteinoids are composed of approximately equal numbers of L- and D-amino acids in contrast to viable proteins with all L-amino acids. Third, there is no evidence that proteinoids differ significantly from a random sequence of amino acids, with little or no catalytic activity. [It is noted, however, that Fox has long disputed this.] Miller and Orgel have made the following observation with regard to Fox's claim that proteinoids resemble proteins:
The degree of nonrandomness in thermal polypeptides so far demonstrated is minute compared to nonrandomness of proteins. It is deceptive, then, to suggest that thermal polypeptides are similar to proteins in their nonrandomness.33
Fourth, the geological conditions indicated are too unreasonable to be taken seriously. As Folsome has commented, "The central question [concerning Fox's proteinoids] is where did all those pure, dry, concentrated, and optically active amino acids come from in the real, abiological world?"34

There is no question that thermal energy flow through the system including the removal of water is accomplishing the thermal entropy and chemical work required to form a polypeptide (300 kcal/mole in our earlier example). The fact that polypeptides are formed is evidence of the work done. It is equally clear that the additional configurational entropy work required to convert an aperiodic unspecified polypeptide into a specified, aperiodic polypeptide which is a functional protein has not been done (159 kcal/mole in our earlier example).

It should be remembered that this 159 kcal/mole of configurational entropy work was calculated assuming the sequencing of the amino acids was the only additional work to be done. Yet the experimental results of Temussi et al.,35 indicate that obtaining all Lamino acids from a racemic mixture and getting alpha-linking between the amino acids are quite difficult. This requirement further increases the configurational entropy work needed over that estimated to do the coding work (159 kcal/mole). We may estimate the magnitude of this increase in the configurational entropy work term by returning to our original calculations (eq. 8-7 and 8-8).

In our original calculation for a hypothetical protein of 100 amino acid units, we assumed the amino acids were equally divided among the twenty types. We calculated the number of possible amino acid sequences as follows:

cr = 100! / 5! 5! 5!....5! = 100! / (5!)20 = 1.28 x 10115 (9-3)

If we note that at each site the probability of having an L-amino acid is 50%, and make the generous assumption that there is a 50% probability that a given link will be of the alpha-type observed in true proteins, then the number of ways the system can be arranged in a random chemical reaction is given by

cr = 1.28 x 10115 x 2100 x 299 = 10175 (9-4)

where 2100 refers to the number of additional arrangements possible, given that each site could contain an L- or D-amino acid, and 299 assumes the 99 links between the 100 amino acids in general are equally divided between the natural alpha-links and the unnatural beta-, gamma-, or epsilon-links.

[NOTE Some studies indicate less than 50% alpha-links in peptides formed by reacting random mixtures of amino acids. (P.A. Temussi, L. Paolillo, F.E. Benedetti, and S. Andini, 1976. J. Mol. Evol. 7, 105.)].
The requirements for a biologically functional protein molecule are: (1) all L-amino acids, (2) all alpha-links, and (3) a specified sequence. This being so, the calculation of the configurational entropy of the protein molecule using equation 8-8 is unchanged except that the number of ways the system can be arranged, (cr), is increased from 1.28 x 10115 to 1.0 x 10175 as shown in equations 9-3 and 9-4. We may use the relationships of equations 8-7 and 8-8 but with the number of permutations modified as shown here to find a total configurational entropy work. When we do, we get a total configurational entropy work of 195 kcal/mole, of which 159 kcal/mole is for sequencing and 36 kcal/mole to attain all L-amino acids and all alpha-links. Finally, it should be recognized that Fox and others who use his approach avoid a much larger configurational entropy work term by beginning with only amino acids, i.e., excluding other organic chemicals and thereby eliminating the "selecting work" which is not accounted for in the 195 kcal/mole calculated above.

In summary, undirected thermal energy is only able to do the chemical and thermal entropy work in polypeptide synthesis, but not the coding (or sequencing) portion of the configurational entropy work. Protenoids are just globs of random polymers. That a polymer composed exclusively of amino acids (but without exclusively peptide bonds) was formed is a result of the fact that only amino acids were used in the experiment. Thus, the portion of the configurational entropy work that was done---the selecting work---was accomplished not by natural forces but by illegitimate investigator interference. It is difficult to imagine how one could ever couple random thermal energy flow through the system to do the required configurational entropy work of selecting and sequencing. Finally, this approach is of very questionable geological significance, given the many fortuitous events that are required, as others have noted.

Solar Energy

Direct photochemical (UV) polymerization reactions to form polypeptides and polynucleotides have occasionally been discussed in the literature. The idea is to drive forward the otherwise thermodynamically unfavorable polymerization reaction by allowing solar energy to flow through the aqueous system to do the necessary work. It is worth noting that minor yields of small peptides can be expected to form spontaneously, even though the reaction is unfavorable (see eq. 8-16), but that greater yields of larger peptides can be expected only if energy is somehow coupled to the reaction. Fox and Dose have examined the peptide results of Bahadur and Ranganayaki36 and concluded that UV irradiation did not couple with the reaction. They comment, "The authors do not show that they have done more than accelerate an approach to an unfavorable equilibrium. They may merely have reaffirmed the second law of thermodynamics."37 Other attempts to form polymers directly under the influence of UV light have not been encouraging because of this lack of coupling. Neither the chemical nor the thermal entropy work, and definitely not any configurational entropy work, has been accomplished using solar energy.

Chemical Energy (Energy-Rich Condensing Agents)

Through the use of condensing agents, the energetically unfavorable dipeptide reaction (G1 = + 3000 cal/mole) is made energetically favorable (G3 < 0) by coupling it with a second reaction which is sufficiently favorable energetically (G2 < 0), to offset the energy requirement of the dipeptide reaction:

dipeptide reaction

A - OH + H - B A - B + H20 G1 > 0 (9-5)

condensing agent reaction

C + H20 D G2 < 0 (9-6)

coupled reaction

A - OH + H - B + C A - B + D G3 < 0 (9-7)

As in thermal proteinoid formation, the free water is removed. However, in this case, it is removed by chemical reaction with a suitable poly- condensing agent-one which has a sufficient decrease in Gibbs free energy to drive the reaction forward (i.e., G2 0 and | G2 | |G1 | so that G1 + G2 = G3 0.

Unfortunately, it has proved difficult to find condensing agents work. for these macromolecule syntheses that could have originated on the primitive earth and functioned properly under mild conditions in an aqueous environment.38 Meanwhile, other condensing agents which are not prebiotically significant (e.g., polymetaphosphates) are used in experiments. The plausible cyanide derivative candidates for condensing agents on the early earth hydrolyze readily in aqueous solutions (see Chapter 4). In the process, they do not couple preferentially with the H20 from the condensation-dehydration reaction. Condensing agents observed in living systems today are produced only by living systems, and thus are not prebiotically significant. Moreover, enzyme activity in living systems first activates amino acids and then brings about condensation of these activated species, thus avoiding the problem of indiscriminate reaction with water.

Notice that if we could solve the very significant problems associated with the prebiotic synthesis of polypeptides by using condensing agents, we would still succeed only in polymerizing random polypeptides. Only the chemical and thermal entropy work would be accomplished by an appropriate coupling of the condensing agent to the condensation reaction. There is no reason to believe that condensing agents could have any effect on the selecting or sequencing of the amino acids. Thus, condensing agents are eliminated as a possible means of doing the configurational entropy work of coding a protein or DNA.

Chemical Energy (Energy-Rich Precursors)

Because the formation of even random polypeptides from amino acids is so energetically unfavorable (G = 300 kcal/mole for 100 amino acids), some investigators have attempted to begin with energy-rich precursors such as HCN and form polypeptides directly, a scheme which is "downhill" energetically, i.e., G < 0. There are advantages to such an approach; namely, there is no chemical work to be done since the bonding energy actually decreases as the energy-rich precursors react to form more complex molecules. This decrease in bonding energy will drive the reaction forward, effectively doing the thermal entropy work as well. The fly in the ointment, however, is that the configurational entropy work is enormous in going from simple molecules (e.g., HCN) directly to complex polymers in a single step (without forming intermediate biomonomers).

The stepwise scheme of experiments is to react gases such as methane, ammonia, and carbon dioxide to form amino acids and other compounds and then to react these to form polymers in a subsequent experiment. In these experiments the very considerable selecting-work component of the configurational entropy work is essentially done by the investigator who separates, purifies, and concentrates the amino acids before attempting to polymerize them. Matthews39 and co-workers, however, have undertaken experiments where this intermediate step is missing and the investigator has no opportunity to contribute even obliquely to the success of the experiment by assisting in doing the selecting part of the configurational entropy work. In such experiments-undoubtedly more plausible as true prebiotic simulations-the probability of success is, however, further reduced from the already small probabilities previously mentioned. Using HCN as an energy-rich precursor, and ammonia as a catalyst, Matthews and Moser40 have claimed direct synthesis of a large variety of chemicals under anhydrous conditions. After treating the polymer with water, even peptides are said to be among the products obtained. But as Ferris et al.,41 have shown, the HCN polymer does not release amino acids upon treatment with proteolytic (protein splitting) enzymes; nor does it give a positive biuret reaction (color test for peptides). In short, it is very hard to reconcile these results with a peptidic structure.

Ferris42 and Matthews43 have agreed that direct synthesis of polypeptides has not yet been demonstrated. While some peptide bonds may form directly, it would be quite surprising to find them in significant numbers. Since HCN gives rise to other organic compounds, and various kinds of links are possible, the formation of polypeptides with exclusively alpha-links is most unlikely. Furthermore, no sequencing would be expected from this reaction, which is driven forward and "guided" only by chemical energy.

While we do not believe Matthews or others will be successful in demonstrating a single step synthesis of polypeptides from HCN, this approach does involve the least investigator interference, and thus, represents a very plausible prebiotic simulation experiment. The approach of Fox and others, which involves reacting gases to form many organic compounds, separating out amino acids, purifying, and finally polymerizing them, is more successful because it involves a greater measure of investigator interference. The selecting portion of the configurational entropy work is being supplied by the scientist. Matthew's lack of demonstrable success in producing polypeptides is a predictable indication of the enormity of the problem of prebiotic synthesis when it is not overcome by illegitimate investigator interference.

Mineral Catalysis

A novel synthesis of polypeptides has been reported44 which employs mineral catalysis. An aqueous solution of energy-rich aminoacyl adenylates (rather than amino acids) is used in the presence of certain layered clays such as those known as montmorillonites. Large amounts of the energy-rich reactants are adsorbed both on the surface and between the layers of clay. The catalytic effect of the clay may result primarily from the removal of reactants from the solution by adsorption between the layers of clay. This technique has resulted in polypeptides of up to 50 units or more. Although polymerization definitely occurs in these reactions, the energy-rich aminoacyl adenylate (fig. 9-1) is of very doubtful prebiotic significance per the discussion of competing reactions in Chapter 4. Furthermore, the use of clay with free amino acids will not give a successful synthesis of polypeptides. The energy-rich aminoacyl adenylates lower their chemical or bonding energy as they polymerize, driving the reaction forward, and effectively doing the thermal entropy work as well. The role of the clay is to concentrate the reactants and possibly to catalyze the reactions. Once again, we are left with no apparent means to couple the energy flow, in this case in the form of prebiotically questionable energy-rich precursors, to the configurational entropy work of selecting and sequencing required in the formation of specified aperiodic polypeptides, or proteins.



Figure 9-1.
Aminoacyl adenylate.

Summary of Experimental Results on Prebiotic Synthesis of protein

In summary, we have seen that it is possible to do the thermal entropy work and chemical work necessary to form random polypeptides, e.g., Fox's proteinoids. In no case, though, has anyone been successful in doing the additional configurational entropy work of coding necessary to convert random polypeptides into proteins. Virtually no mechanism with any promise for coupling the random flow of energy through the system to do this very specific work has come to light. The prebiotic plausibility of the successful synthesis of polypeptides must be questioned because of the considerable configurational entropy work of selecting done by the investigator prior to the polymer synthesis. Surely no suggestion is forthcoming that the right composition of just the subset of amino acids found in living things was "selected" by natural means, or that this subset consists only of L-a-amino acids. This is precisely why a large measure of the credit in forming proteinoids must go to Fox and others rather than nature.

Summary of Experimental Results on Prebiotic Synthesis of DNA

The prebiotic synthesis of DNA has proved to be even more difficult than that of protein. The problems that beset protein synthesis apply with greater force to DNA synthesis. Energy flow through the system may cause the nucleotides to chemically react and form a polymer chain, but it is very difficult to get them to attach themselves together in a specified way. For example, 3' - 5' links on the sugar are necessary for the DNA to form a helical structure (see fig. 9-2). Yet 2'-5' links predominate in most prebiotic simulation experiments.45 The sequencing of the bases in DNA is also crucial, as is the amino acid sequence in proteins. Both of these requirements are problems in doing the configurational entropy work. It is one thing to get molecules to chemically react; it is quite another to get them to link up in the right arrangement. To date, researchers have only succeeded in making oligonucleotides, or relatively short chains of nucleotides, with neither consistent 3'-5' links nor specific base sequencing.



Figure 9-2.
A section from a DNA chain showing the sequence AGCT.

Miller and Orgel summarized their chapter on prebiotic condensation reactions by saying:

This chapter has probably been confusing to the reader. We believe that is because of the limited progress that has been made in the study of prebiotic condensation. Many interesting scraps of information are available, but no correct pathways have yet been discovered.46
The situation is much the same today.

Summary Discussion of Experimental Results

There is an impressive contrast between the considerable success in synthesizing amino acids and the consistent failure to synthesize protein and DNA. We believe the reason is the large difference in the magnitude of the configurational entropy work required. Amino acids are quite simple compared to protein, and one might reasonably expect to get some yield of amino acids, even where the chemical reactions that occur do so in a rather random fashion. The same approach will obviously be far less successful in reproducing complex protein and DNA molecules where the configurational entropy work term is a nontrivial portion of the whole. Coupling the energy flow through the system to do the chemical and thermal entropy work is much easier than doing the configurational entropy work. The uniform failure in literally thousands of experimental attempts to synthesize protein or DNA under even questionable prebiotic conditions is a monument to the difficulty in achieving a high degree of information content, or specified complexity from the undirected flow of energy through a system.

We must not forget that the total work to create a living system goes far beyond the work to create DNA and protein discussed in this chapter. As we stated before, a minimum of 20-40 proteins as well as DNA and RNA are required to make even a simple replicating system. The lack of known energy-coupling means to do the configurational entropy work required to make DNA and protein is many times more crucial in making a living system. As a result, appeals to chance for this most difficult problem still appear in the literature in spite of the fact that calculations give staggeringly low probabilities, even on the scale of 5 billion years. Either the work---especially the organizational work---was coupled to the flow of energy in some way not yet understood, or else it truly was a miracle.

Summary of Thermodynamics Discussion

Throughout Chapters 7-9 we have analyzed the problems of complexity and the origin of life from a thermodynamic point of view. Our reason for doing this is the common notion in the scientific literature today on the origin of life that an open system with energy and mass flow is a priori a sufficient explanation for the complexity of life. We have examined the validity of such an open and constrained system. We found it to be a reasonable explanation for doing the chemical and thermal entropy work, but clearly inadequate to account for the configurational entropy work of coding (not to mention the sorting and selecting work). We have noted the need for some sort of coupling mechanism. Without it, there is no way to convert the negative entropy associated with energy flow into negative entropy associated with configurational entropy and the corresponding information. Is it reasonable to believe such a "hidden" coupling mechanism will be found in the future that can play this crucial role of a template, metabolic motor, etc., directing the flow of energy in such a way as to create new information?

References

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19. G. Nicolis and I. Prigogine, 1977. Self Organization in Nonequilibrium Systems. New York: Wiley.

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22. Prigogine, Nicolis, and Babloyantz, Physics Today, p.23-31.

23. Ibid; Nicolis and Prigogine, Self Organization in Nonequilibrium Systems.

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25. P.T. Mora, 1965. In The Origins of Prebiological Systems and of Their Molecular Matrices, p.39.

26. E.R. Harrison, 1969. In Hierarchical Structures. ed. L.L. Whyte, A.G. Wilson, and D. Wilson, New York: Elsevier, p.87.

27. Nicolis and Prigogine, Self Organization in Nonequilibrium Systems.

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29. Kaplan, Rad. Environ. Biophysics, p.31.

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34. C.E. Folsome, 1979. The Origin of Life. San Francisco: W.H. Freeman, p.87.

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38. J. Hulshof and C. Ponnamperuma, 1976. Origins of Life 7, 197.

39. C.N. Matthews and R.E. Moser, 1966. Proc. Nat. Acad. Sci. U.S. 56, 1087; C.N. Matthews, 1975. Origins of Life 6, 155; C. Matthews, J. Nelson, P. Varma, and R. Minard, 1977. Science 198 622; C.N. Matthews, 1982. Origins of Life 12, 281.

40. C.N. Matthews, and R.E. Moser, 1967. Nature 215,1230.

41. J.P. Ferris, D.B. Donner, and A.P. Lobo, 1973. J. Mol Biol. 74, 499.

42. J.P. Ferris, 1979. Science 203, 1135.

43. C.N. Matthews, 1979. Science 203, 1136.

44. Katchalsky, Die Naturwiss., p.215.

45. R.E. Dickerson, September 1978. Sci. Amer., p.70.

46. Miller and Orgel, The Origins of Life on the Earth, p.148.

Chapter 8

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