Science in Christian Perspective

Biochemical Complexity 
and Its Significance In Evolution*

WALTER R. HEARN, Ph.D.

Assistant Professor of Biochemistry

Baylor University College of Medicine

Houston 25, Texas

I should like to begin my first paper before the American Scientific Affiliation with that classical definition of a biochemist which differentiates him from other species of the genus Chemist. According to this epistemological trilogy, a physical chemist is a chemist who makes very accurate measurements of the properties of very impure substances; an organic chemist is one who makes very inaccurate measurements on extremely pure compounds; and a biochemist is someone who makes the sloppiest kind of measurements on the crudest sort of materials. This would be embarrassingly accurate were it not for the fact that most biochemists are educationally heterozygous, and carry in their genetic make-up either a dominant or recessive trait for the more exact and quantitative branches of chemistry. This accounts for the healthy and vigorous condition of the breed today; i.e., we are purifying the materials we study, and our measurements are becoming more significant. This has allowed a substantial theoretical framework to be established, or in other words, has allowed biochemistry to take its: place as a science, capable not only of further development itself, but also of making an impact on other disciplines and of influencing their course of development.

It is this impact of biochemistry on evolutionary hypotheses which I am discussing today. Whatever our theory of the mechanism of variation among living things, we must all agree that in the last analysis (or perhaps the next-to-last!) such variation either is caused by or results in changes in the arrangement of specific atoms in specific molecules in the living cell. J. B. S. Haldane was essentially correct when he said, "Our final theory of evolution will see it largely as a biochemical process" (1). That is to say that the predominant theme in evolutionary considerations in the future will be metabolism rather than morphology (2).

The biochemical information on which such a structure must be built has come slowly, but the rate at which it is accumulating is now accelerating quite rapidly. For generations chemists have been isolating and purifying the products of metabolism of the widest

*Paper presented as part of a symposium on Entropy and Evolution at the Ninth Annual Convention of the American Scientific Affiliation at Harrisonburg, Virginia, August 24-26, 1954.

variety of living organisms. As you would expect, compounds which are relatively stable, or in other words, compounds of low levels of chemical energy, were the ones which were first isolated and studied, in contrast to more complex, less stable molecules we shall discuss later on.

Even though a number of such stable "natural products" have been known in reasonably pure form for a very long time, it is only in modern times that analytical and synthetic methods have been perfected for the determination of their structure. For example, crystalline cane sugar was known and used as early as 300 A.D. (3), but its chemical structure was not established by degradation definitely until around 1927, its in vitro enzymatic synthesis not accomplished until 1944 (4), and its unequivocal chemical synthesis only very recently reported (5). The first amino acid to be isolated was a relatively simple molecule, cystine, which Wollaston obtained from urinary calculi in 1810; but it was not until 1846 that a correct analysis was reported, and not until 1904 that its structure was finally established (6). A characteristic material from opium was first crystallized as early as 1803, and morphine was isolate~ and recognized as an alkaloid in 1814 (7), but its final proof of structure by total synthesis was only recently accomplished (8). The "unorthodox" and unexpected structural features of some of the antibiotics produced by lowly micro-organisms are having a profound effect on modern organic chemical theory (9).

The structures of hundreds or even thousands of chemical compounds produced by living organisms are now known with certainty. Some morphologist, resenting the obsolescence to which I have assigned him in a previous paragraph, may care to point out that all of this structure business is really just chemical morphology, which is certainly true; but the understanding of such chemical structures is only the starting point of modern dynamic biochemistry, whose goal is the understanding of the reactions by which such compounds are produced. Biochemists in the past two decades using such penetrating and precise methods as the isotopic tracer technique, have elucidated the chemical steps by which many compounds are produced biologically; as can be seen from the chapters on "Intermediary Metabolism" in any up-to-date textbook of biochemistry (10), or the very readable book on metabolism by Ernest Baldwin of Cambridge University (11). And now, without going into detail, I should like to mention three areas of investigation in which our understanding of these metabolic pathways is having an influence on evolutionary thought.

The first of these is COMPARATIVE BIOCHEMISTRY. Comparative biochemistry deals with biochemical relationships between different species . Biochemists have always made use of a variety of certain types of organisms offer specific advantages for each type of investigation. However, a relatively insignificant number of the thousands of species known have been studied biochemically at all, and of these an even smaller number have been studied thoroughly. In recent years attempts have been made to investigate certain aspects of the metabolism of representatives of at least the major genera. Studies of nutritional requirements, nitrogen excretion, oxygen transport, etc., have been made in a reasonably systematic manner. Modern microchemical and particularly chromatographic methods are yielding quantitative information of genuine value. At the present time, classification of species along biochemical lines more or less parallels the classifications previously made along morphological lines (1) ; or to boil it down to the simplest terms, animals which look alike, are alike biochemically, and animals which appear to be different, are different. Some of the difficulties encountered in such studies are the variability among different organisms within the same species (emphasized recently with regard to human beings by R. J. Williams (12), the manipulation of the smaller species to obtain sufficient quantities of their tissues, organs, or secretions for analysis, and the decision as to what constitute the really significant properties for comparison; similar problems are inherent in any kind of attempt at biological classification. A very satisfactory introduction to this field is provided by another very readable little book by Ernest Baldwin (13).

The second area is BIOCHEMICAL GENETICS, which deals with metabolic differences among subsequent generations within a given species. The finding that certain mutants of micro-organisms are unable to carry out specific metabolic reactions has made microbiology a valuable tool in the study of metabolic pathways. Beadle discovered in 1941 (14) that such "biochemical mutants" of the mold Neurospora crassa could be produced by artificial means such as irradiation, and the theory soon developed that a single gene may control the production of a single enzyme and thus be responsible for a single step in a chain of metabolic reactions. But biochemical investigations on higher plants and animals and also on man have their influence on genetics. Itano's discovery of 
an electrophoretically abnormal hemoglobin in people with sickle cell anemia, and subsequent investigations in this field 05) have led to the concept of inherited "molecular disease." Recent evidence that the sickle cell trait may also carry immunity to malaria (16) provides a possible explanation for the "very thorny problem in gene dynamics" of the high incidence among Negroes of hereditary anemias (17). This cross-fertilization of genetics and biochemistry provides a penetrating tool for the investigation of some of the basic problems at the heart of evolutionary considerations.

A third area of biochemical inquiry, which this time deals with the relationship of the metabolism of various parts of the same organism, is that of BIOCHEMICAL EMBRYOLOGY. Work in this field is aimed at unraveling the mechanisms by which individual cells achieve metabolic individuality, and thus produce tissues and organs of specialized form and function. Joseph Needham's encyclopedic works (18) have merely set the stage for modern expansion in this field. The Proceedings of a Symposium on the Biological and Structural Basis of Morphogenesis have recently been published as a supplement to a Dutch journal (19).

In spite of rapid developments in each of these areas, one gets the impression from reading even the most current literature that only the groundwork has been laid; and that the complexity of living things revealed by such studies leaves us face to face with more perplexing problems than the ones we started out to solve. Comparative biochemistry has shown, for example, that even the "simplest" forms of life have extremely complex metabolic patterns, and that in spite of early popular enthusiasm about viruses, there exists a tremendous gap between the living and the non-living. Biochemical genetics leaves us puzzled before the combination of stability and variability which the genic apparatus must possess. Biochemical embryology staggers us with the concept of the entire metabolic potentiality of an organism, packed into a single fertilized ovum. Dr. Sam Granick of the Rockefeller Institute for Medical Research, after discussing the steps involved in the synthesis of hemoglobin in the animal body at a conference on protein metabolism a year ago, added this comment:

"From the little that we know and the much more that we have hypothesized, we have obtained a glimpse into a highly intricate pattern of great exactness. At the same time, one conclusion is forced upon us. It is that this hypothetical slime-stuff which we call protoplasm must be endowed with such infinitely complicated systems and such delicate interrelations that only by the most distant stretch of the imagination could it possibly be postulated to exist at all!" (20)

It is in revealing this "complexity," or "delicateness," or "intricacy," or "orderedness," or "low entropy" of the living stuff, that biochemistry has its influence on evolutionary theory. For, the more complex we discover life to be, the more difficult it is to account for it. If we assume that the first living things were far simpler than those we study today, the problem of increasing complexity, coupled with stability, confronts us. If on the other hand we feel that the primitive living stuff had much in common with life today, the problem of its origin from inanimate matter becomes even more staggering. There is probably a tendency in this latter direction of thought today (21, 21a), that is, to consider that there was a tremendous accumulation of free energy in the form of organic compounds at the time of origin of primeval life. There has been considerable enthusiasm among those who hold this view over the results of an experiment reported last year (22), in which some of the naturally occurring amino acids were produced by the continuous cycling of a mixture of methane, ammonia, hydrogen, and water vapor through a high-voltage arc (21a).

However, the amount of enthusiasm over a scientific discovery is not necessarily a measure of its significance. In 1907, when Emil Fischer succeeded in combining eighteen amino acid molecules in a chemically controlled manner to form a peptide of molecular weight greater than 1,200, excited reporters announced to the world that the "greatest riddle of life" had been solved (23). Fischer knew and emphasized, however, that in spite of the importance of his accomplishment, we were still a long way off from synthesizing even the simplest of naturally occurring proteins.

Synthetic methods have also been developing rapidly, and duVigneaud and his coworkers last year were able to announce the synthesis of the octapeptide hormone of the posterior pituitary, ocytocin, almost simultaneously with the announcement of its structure (29). The controlled synthesis of polypeptides of specific amino acid sequence is still a laborious and sometimes tedious job, but the synthesis of such a polypeptide as ACTH. or of a protein such as insulin is certainly not out of the question in the near future.

The protein structures which intrigue us most, however, are those with which is associated the ability to catalyze specific chemical reactions, i.e., the enzymes. Our knowledge of these structures is very meager at the present time. For one thing, the enzymes are generally of considerably higher molecular weights than that of insulin; they are rather easily denatured or inactivated, and are difficult to obtain as absolutely homogenous substances. A total of approximately 72 enzymes have been crystallized to date (30), but crystalinity does not necessarily imply homogeneity in the case of complex molecules such as proteins. Where "co-enzymes," molecules much smaller and less complex than proteins, are involved as the active sites on the enzyme molecules, it has been generally possible to propose a step-wise mechanism for the enzymatic reaction, based on the chemical structure of the coenzyme; certain similarities in the pattern of such reactions have emerged (31). But the real puzzlers are the enzymes which apparently contain no prosthetic group apart from their specific amino acid configuration, and here our understanding must await the elucidation of the details of their fine structu~re. This information should accumulate in the near future.

The enzymatic mechanisms which are of the greatest interest to us here today are those involved in the biosynthesis of proteins, and specifically, the biosynthesis of the enzymes themselves. For it is the ability of the organism to synthesize enzymes which must be the essence of its ability to reproduce itself. Forming the peptide bonds in a protein is an "endergonic" process; i.e., it requires free energy. This free energy must come from "exergonic," or energy-yielding, processes, and one of the most absorbing problems of biochemistry today is finding out how these processes are coupled.

Most of these investigations involve "model" systems, much simpler than anything that goes on inside the cell. One of these is the "transpeptidation" system of J. S. Fruton and others, with which I have had some slight experience. In such a system, the amount of energy necessary is cut down by using starting materials already containing amide bonds and allowing the enzyme to catalyze a "transamidation" rather than the de novo formation of an amide bond; the energy is supplied by the insolubility of the polypeptide product, which in this case comes out of solution, driving the reaction to completion. With such a system it was possible to produce a peptide chain containing eight amino acid residues, starting from glycyl-Lphenylalanine amide and using a purified protease from beef spleen as the enzyme (32). One difficulty in the concept that protein biosynthesis is merely a "reversal of proteolysis" catalyzed by proteases is the high degree of specificity oi the known proteases; in other words, there would almost have to be a separate enzyme for each bond in the protein synthesized, which seems unlikely. Fox at Iowa State has shown that the substrate itself may have an ef f ect on the choice of possible synthetic reactions catalyzed by a protease (33), and has thus partially overcome this difficulty.

A concept of protein biosynthesis perhaps more favored than the "reversal of proteolysis" idea is what may be called the "template," or "zipper' theory. According to this theory, the amino acids line up in the proper order along a "template," and a reaction occurs by which the peptide bonds are all formed essentially simultaneously. Lipmann, using the synthesis of pantoylL,,B-alanine as a model (34), has suggested a possible mechanism for the reaction in which the peptide bonds are formed, utilizing indirectly the high energy bonds of ATP (30. A comprehensive review of various lines of investigation in the fields of peptide bond formation has been provided recently b,y Bo~rsook (3,5).

To sum up, just where do we stand in our efforts to understand what actually goes on in living things? It is perhaps appropriate to repeat here Krebs' words of caution about the interpretation of in vitro experiments:

"Physiologically most intermediates exist only transitorily, i.e., in minute quantities. Moreover, they occur only intracellulary. These circumstances preclude their identification under 'physiological' conditions. To investigate intermediary metabolism, the concentration of the metabolite must be artificially raised, or poisons must be added, and/or the tissue has to be removed from its normal site and to be perfused, or sliced, or minced, or extracted. The statement, therefore, that the evidence is valid for living tissues under 'physiological' conditions always implies the assumption that the reactions occur under conditions different from those of the experiment. As far as one can see, this state of affairs is bound to persist, and for this reason the theory of intermediary reaction mechanism is bound always to remain a theory.

In short, while it can be proved that a tissue or a cell has the ability to perform certain reactions, the ,physiological' occurrence of the reactions must remain an assumption. If one agrees with Hopkins' contention that, in general, a tissue is able to deal only with what is customary to it, the demonstration of an intermediary reaction under experimental conditions can be regarded as powerful evidence, though not a final proof, that the reaction is part of the normal metabolism of the tissue." (36).

But assuming that our experiments do tell us what is actually going on inside the cell, are we now in a better position to explain the origin and variations of this living stuff? Personally, I think we are not. Our concept of protoplasm is no longer the naive one of Darwin's day. We see in the living cell dozens, or hundreds, or even thousands of intricate protein molecules, delicately arranged, yet in a dynamic equilibrium in which this extremely low level of entropy is maintained by the expenditure of free energy in metabolic reactions (37). Some of these reactions we say we understand; yet we are completely unable to account for the existence of the system which makes them possible.

At least that is what I hope to do.

Reference

(1) Florkin, M., Biochemical Evolution. Ed., tr., and augm. by S. Morgulis New York: Academic Press 1949.

(2) Blum, H. F. Time's Arrow and Evolution. Princeton: Princeton University Press, 1951.

(3) Pigman, W. W., and R. M. Goepp, Jr., Chemistry of the Carbohydrates. New York: Academic Press, 1948. p. 1, pp. 450-451.

(4) Hassid, W. Z., M. Doudoroff, and H. A. Barker, "Enzymatically Synthesized Crystalline Sucrose." J. Ant. Chem Soc. 66, 1416 (1944).

(5) Lemieux, R. U., and G. Huber, "A Chemical Synthesis of Sucrose." J. Am. Chem. Sec. 75, 4118 (1953).

(6) Vickery, H. B., and C. L. A. Schmidt, "The History of the Discovery of the Amino Acids." Chem. Revs. 9, 169318 (1931).

(7) Farber, E., "Bio-active Substances in the Nineteenth Century." Chymia 3, 63-76 (1950).

,(8) Gates, M., and G. Tschudi, "The Synthesis of Morphine." J. Am. Chem. Soc. 74, ll()9-10 (1952).

Cram, D. J., "Antibiotics and Chemical Orthodoxy." Chem. and Engin. News 32, 1120-1 (1954).

,(10) Such as Fruton & Simmonds, General Biochemistry, New York: Wiley, 1953; or West & Todd, Textbook of Biochemistry, New York: The Macmillan Co., 1951.

(11) Baldwin, E. Dynamic Aspects of Biochemistry. Cam bridge: The University Press, 2nd Edn., 1952.
(12) Williams, R. J., Free and Unequal. Austin: University of Texas Press, 1953.
(13) Baldwin, E., An Introduction to Comparative Biochemistry. Cambridge: The University Press, 3rd Edn., 1948.

(14) Beadle, G. W., and E. L. Tatum, "Genetic Control of Biochemical Reactions in Neurospora." Proc. Natl. Acad. Sci. 27, 499-506 (1941) ; Beadle, G. W., "Biochemical Genetics," Chem. Revs. 37, 15-96 (1945).

(15) Itano, H. A., "Human Hemoglobin." Science 117, 89-94 (1953).

(16) Science 119, 718 (1954) ; Allison, A. C., "Protection Afforded by Sickle-Cell Trait Against Subtertian Malarial Infection," British Med. J. (No. 4857) 290-4 (Feb. 6, 1954).

(17) Neel, J. V., "The Population Genetics of Two Inherited Blood Dyscrasias in Man," Cold Spring Harbor Symposia on Quant. Biol. 15, 141-58 (1950).

(18) Needham, J., Chemical Embryology, 3 Vols. Cambridge: The University Press, 1931; Biochemistry and Morphogenesis, Cambridge: The University Press, 1942.

(19) Arch. neerl. zool. 10, Supplement 1 (1953). cf. Chein. Abstracts 48, 6479b (1954).

(20) "C. & E. N. Reports: Conference on Protein Metabolism," Chem. and Engin. News 31, 668 (1953).

(21) Oparin, A. L, The Origin of Life. Tr. and augm. by S. Morgulis. New York: Dover Publications, 2nd Edn., 1953.

(22) Miller, S. L., "A Production of Amino Acids Under Possible Primitive Earth Conditions," Science 117, 528-9 (1953) ; cf. "Genesis by Lightning," Scientific American 189 (No. 1) 42-4 (July, 1953).

(24) Moore, S., and W. 1-1. Stein, "Photometric Ninhydrin Method for Use in the Chromatography of Amino Acids," J. Biol. Chem. 176, 367-88 (148) ; "Chromatography of Amino Acids on Sulfonated Polystyrene Resins," Ibid., 192, 663-81 (1951).

(26) Desnuelle, P., "Quelques Techniques Nouvelles pour I'Etude de la Structure des Proteines," Advances in Enzymol., 14, 261 (1953).

(27) Fraenkel-Conrat, fl., "A Technique for Stepwise Degradation of Proteins from the Amino End," J. Ant. Chem. Soc. 76, 3606 (1954)

(28) Pauling, L., R. B. Corey, & R. Howard, "The Structure of Protein Molecules," Scientific American, 191 (No. 1) 51-9 (July, 1954).

(29) duVigneaud, V., C. Ressler & S. Trippett, "The Sequence of Amino Acids in Oxytocin, With a Proposal for the Structure of Oxytocin," J. Biol. Chem. 205, 949 (1953) ; duVigneaud, et al., "The Synthesis of an Octapeptide Amide with the Hormonal Activity of Oxytocin," J. Am. Chem. Soc. 75, 4879 (1953).

30) Schwimmer, S., and A. B. Pardee, "Principles and Procedures in the isolation of Enzymes," Advances in Enzymol. 14, 375 (1953).

(31) McElroy, W. D., and B. Glass, A Syinposium on the Mechanism of En~yvie Action. Baltimore: The Johns Hopkins Press, 1954. Summary by Bentley Glass, pp. 740-805.

(32) Fruton, J. S., W. R. Hearn, V. Al. Ingram, D. S. Wiggans, and M. Winitz, "Synthesis of Polymeric Peptides in Protease-Catalyzed Transamidation Reactions,' J. Biol. Chem. 204, 891-902 (1953).

(33) Fox, S. W., M. Winitz, and C. W. Pettinga, "Enzymatic Synthesis of Peptide Bonds. VI. The Influence of Residue Type on Papain-Catalyzed Reactions of Some Benzoylamino Acids with Some Amino Acid Anilides," J. Am. Chem. Soc. 75, 5539 (1953).

(34) Mass, W. K., and G. D. Novelli, "Synthesis of Pantotbenic Acid by Depyrophosporylation cf Adenosine Triphosphate," Arch. Biochem. and Biophys. 43, 236-8 (1953).

(35) Borsook, H., "Peptide Bond Formation," Advances in Protein Chem. 8, 128-74 (1953) ; Vor a brief discussion see: Linderstrom -Lang, K. U., "How is a Protein Made?" Scientific American 189 (No. 3) 100-6 (Sept., 1953).

(36) Krebs, H. A., "The Intermediar- Stages in the Biological Oxidation of Carbohydrate," Advances in Enzyniol. 3, 191-252 (1943). Footnot,- on p. 220.

(37) Gutfreund, H., "The Nature of Entropy and its Role in Biochemical Processes," Advances in, Enzymol. I 1-33 (1951).

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