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Energetics of Polymerization A Contribution to an Understanding of Protein Synthesis Herbert C. Friedmarin Department of Biochemistry and Molecular Blology, University of Chicago, Chicago, IL 60637
Current textbook treatments of the energetics of protein synthesis show a variety of approaches, unmatched by presentations of anv other biochemical topic. Frequently one finds straightfo;ward descriptions of the various processes that require the breakdown of ATP or of GTP (14). In many instances a GTP-utilizing process is discussed from an assortment of viewpoints such as cyclic association and release of elongation factors (5), directed movement (5, 6), conformational effects (7-9), and proofreading (8).A few books express ignorance and wonder, as when one reads T h e molecular processes that underlie protein synthesis. . . do not make co&eptual sense in the way that DNA transcription, DNA repair, and DNA replication do. . . Thus the details of orotein svnthesis must lareelv - be learned as fact without an obvious conceptual framework" (lo), or "Generally when [GTP and ATP] are hydrolyzed, the free energy of hydrolysis is used to drive reactions that otherwise are enereeticallv unfavorable. This does not seem to be the case in protein-synthesis" (9). A more biological approach points out that the impressive free energy expenditure for peptide bond hiosynthesis is the price paid for nearly perfect fidelity in biological translation (11, IZ), but the manner in which fidelity and energy expenditure are coupled is not discussed. In another textbook the energetic requirements for protein synthesis are spelled out explicitly with the statement that protein synthesis addresses itself to various chemical problems that include "to overcome thermodynamic barriers. . . and . . . to establish the pattern or sequence in which the monomer units are linked toaether" (13). Here again the . various energy-requiring steps are not treated in terms of their individual contributions to the overlapping dictates of thermodynamics and of amino acid ordering. In every textbook a description of the individual steps in protein synthesis obscures an overall energetic rationale. It is remarkable that the energetics of the one process that consumes far more biosyntbeticknergy of an organism than any other (up to 90%)should still be so mysterious. An attempt will be made here to reach a possibly more constructive approach to the energetics of protein synthesis by emphasizing the ordering a s ~ e i t of s thiorocess. Primary protein structure, i.e., the immediate product of nrotein svnthesis. manifests two degrees of order when compared to the disorder of amino acid mixtures: the first degree of order accrues from the ioinine of amino acids by means of peptide bonds, the second degree from joining in predetermined sequence. One may distinguish these two orders in terms of the randomness bf the &ray of linked monomers. The ordering of amino acids in a fixed sequence represents a much higbe; degree of unshuffling than does random covalent ioininp. It is not immediately apparent how the energy requirements for these two degrees ordering can be compared numerically, particularly since no biological energyieouirine exists that favors the ioinine of randomlv " svstem " arrayed amino acids. (The reverse of proteolysis is not favored enereeticallv and hence need not be considered here.) It is, therefore, ofinterest that in the closely related area of nucleic acid biosynthesis a test case is available that does
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permit one to compare the energetics of a random versus an ordered ~olvmerizationof monomers. Two comoletelv dif~-~~~~~~~ ferent types of enzymes exist that catalyze the formation of RNA. Ordered RNA is made in the oresence of templaterequiring RNA polymerase, while raniomly ordered RNA is made by polynucleotide phosphorylase. The polymerase uses a nucleoside triphosphate per nucleoside monophosphate incorporated, while the phospborylase uses a nucleoside diphosphate per incorporated monophosphate. It is usually stated that a greater "pull" occurs in a reaction in which inorganic pyrophosphate is a product than in a reaction where inorganic orthophosphate is formed, since the pyrophosphate can itself bi hyd;olyzed to orthophosphate. 15neraetically,the conversion of a nucleoside triphusphate co a nucieosidemonopbosphate is equivalent to the conversion of two nucleoside triphosphates to nucleoside diphosphates. In the present case the reaction catalyzed by the polymerase is twice as "expensive" as that catalyzed by the phosphorylase. However. when one stresses ordering rather than " ~ u l l " a comparison between these two reaction types is very revealine: the conclusion is inescapable that the different energy expenditures in these two reartions (i.e., the utilization of one or of two"hiah enerrv" phuiphate bonds), are related to the degree of or>er of the prod&ts. It is also clear that here extra "pull" is associated with extra "ordering". The analogy to the two levels of amino acid ordering is obvious. The standard free energy of hydrolysis of a peptide bond, about -5.5 kcallmol, is very similar to that of a nucleotide to a nucleoside,about -3.4 kcallmol. Since in protein synthesis the formation of a oeotide . . bond between ordered amino acids requires the equivalent of the conversion of four nucleit follows oside triohosohates to nucleoside diohosohates. . . . . that, by analogy with the energetics of ordered v&~s unordered nucleotide polymerization, possibly as much as threefourths of the energy utilized in protein synthesis contributes to the ordering of the individual amino acids. Why, then, is the process of amino acid orderingin protein synthesis so much more expensive than that of nucleotide ordering? The answer must be related to the fact that the degree of orderina or of unscrambling associated with the selection of one outof 20 amino acids & greater than that associated with the selection of one out of four nucleotides. In the absence of systems that produce polymers of unordered amino acids, it is instructive to turn to the energetics of simple peptide bond-forming reactions. Although the comparison of these reactions to ribosome-dependent protein synthesis is not as elegant as the comparison of polynucleotide phosphorylase with RNA polymerase, the conclusion reached is aeain - compelling: - In some instances where theordering process isnot aprimary concern only one "highenerev" phosphate bond is expended per peptide bond formed. Thus the activity of glut&nine sy&b$aie, resulting in the formation of an unsubstituted amide bond, is associated with the degradation of just one ATP to ADP (14-16). Perhaps more to the point, each step in the formation of the two peptide bonds of glutathione, catalyzed by two successive enzymes, is associated with the expenditure of only one
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"high-energy" phosphate bond (17-29). In the case of the formation of gramicidin S a n d other peptide antibiotics one ATP is converted to AMP per peptide bond formed (20), a process energetically equivalent to the expenditure of two "high-energy" phosphate bonds. Here, without going into details, the enzyme takes on a template-like function, and one can say that a certain selection procesi is necessarily associuted with a degree uf ordering higher than in the case of glutathione formation but lower than in the case of larger proteins. I t has been suggested that peptide antibiotic synthesis, which uses protein templates.. mav" be reearded as an evolutionary step towards ~ R N and A ribosome-dependent protein synthesis (21,22). One must hasten to add that there are well recognized instances of simple peptide bond formation aisociated u,ith the expenditure of two "hirh-enerm" honds per peptide bond rather than one, as for instancesin the synthesis of hippuric acid (N-benzoylglycine) (23, 24), carnosine (N-8-alanyl-L-histidine),and anserine (N-8-alanyl-3-methyl-I-histidine) (25-27). I t is clear that in these cases, all of which involve adenylate intermediates, the primary concern is one of energetic "pull". An attempt can now he made to see how the individual steps in protein synthesis (i.e., excluding initiation and termination, which are energeticallv insianificant in the overall scheme) can be seen to c&tribuie to ;ach of the two degrees of order in the product. In biological thinkine one is constantly challenged to make correlations betwein biochemical reactions and the resulting physiological processes, i.e., between events at the molecular level and a t the organismic level. One of the challenges provided by the existence of protein synthesis is that here it is necessary to make correlations between molecular event and physiological function a t the cellular level. Amino acid activation, which utilizes the equivalent of two "high-energy" bonds, and which is usually regarded, in analom to fattv acid activation. exclnsivelv as the energizing steppreparatory to peptide bbnd formacon, necessarily includes contributions to the second degree of order, since the specific relationship between amino acid and tRNA requires a selection by the activating enzvme of one among 20 amino acids a n d o n e among abbut 32 tRNA's. Again, a t the beginning of chain elongation, the addition of aminoacyl-tRNA to the ribosomal system as dictated by mRNA codons constitutes a selection among various available charged tRNA's. On the other hand, a t the end of chain elongation, the process of translocation does not appear to include selection since the amino acids have already been joined. The conclusion, that three of the four "high-energy" bond equivalents utilized in protein svnthesis contribute to the second degree of amino &id ordeiing, is ronsistent with that reached when the energetics of protein synthesis and of (In the ordered and unord~.redRNA sgnthrsisarecom~~ared. above approach the energetirs of formation of the numerous participants in protein synthesis were ignored; similarly, the energetics of formation of DNA templates were ignored when the ordered RNA formation was considered.) A fascinating question concerns one aspect of the apparent lack of conceptual sense of the process of protein synthesis (9, 10) that is not encountered in the study of other biosynthetic processes. This question suggests itself in vari-
ous ways, all of which appear to assume a conflict with thermodynamic intuition:.^^ pathways determine energetics? Are the energetics of pathways dictated by their complexity? The answer must of course he that energetics comes first. I t follows that the energetics of ordered protein svnthesis require the utilization-by whatever mechanismiof the equivalent of four "high-energy" phosphate honds, so that mechanistically a system had to he elahorated that satisfied these pre-existing energetic requirements. This conclusion is reached quite independently of the circumstance that no theoretical prediction can yet he made of the energetic requirements for ordered protein svnthesis. In other words. the pathway selected do& not determine the energetics of the observed mechanism; it is simply . . consistent with the energetics. Obviously, when ordering is a part of an energetically favored chemical reaction, i t is quite impossible to dissect ordering from "pulling". In the case of a condensation reaction such as the one catalyzed by RNA polvmerase, h i fact is easily appreciated since lust unp ene;gylcoupled reaction is involved per condensation. In the rase of prutein synthesis, however. the matter is far more comdei., since ~~~fkom thr energetic requiremenr. that the equivalent of as many ar; four "high-enwgv" phusphate honds be utilized for each ordered ct~ndensa~inn, it follows that merhanisticallg the participation of compounds such as ATP or ( X I ' ran be parcelled out or quantized over different steps of the proceis. This kind of approach to protein synthe~isclarifies some of the energetic cnn~indrumspmrd hy this nece.isarily complex system, and also inwrporates some of its derails into a conceptual framework
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Literature Clted (1) McGilvcry, R. W . '"Biochemistry, a Fundionsl Approach".3rd ed.; Ssuodcrs: Philadelphia, 1983: pp 92-99. (2) Newsholmc, E. A,; Leach, A. R. "Biochemistry for the Medical Sciences"; wi1ey: Chiehester. 1983: pp 66L673. (3) Rawn,J. D. "Biochemistry"; Harper & Row: New Yark, 1988;pp 106%1072. (4) Smith. E. L.: Hill, R. L.: Lehman, I. R.: LefLoxitz. R. J.; Handler, P.: White, A. "Principles of Biochcmirtry': 7th ed.; McGrew-Hill: New York. 1983; pp 735.736. (5) Stryer. L. "Biochemistry", 2nd ed.; Freeman. San Francisco,1981;pp659-660 161 Stenf, G. S.; Catendax, R. "Molecular Genetics. An Introductory Narrative": 2nded.: Freeman: Ssn Francisco, 1978;p 528. (7) Wataan. J. D. "Molecular Biology of the Gmc". 3rd ed.: Benjamin: Medo Park. CA, 1976: pp 337-338. (81 Balky. J. W. In "Biochemistry":Zubay, G., Caord. Author; Addison-Weaiey: Reading, MA, 1983: pp 948-956. (91 Freihlder, D. '"Molecular Biology": Van Nostrand Reinhold: New Yark, 1983:p 517. (10) Alberta, B.; Bray, D.: Lewis, J.; Raff, M.; Roberta, K.: Wslson, J. D. "Molecula. Bioiagy of the Cell": Garland: Now York, 1983: p 202. (11) Lehninger, A. L. "Biochemistry":2nd ed.: Worth: New York, 1975: p 949. (12) Lahninger, A. L:'Prineiples of Biochomistr/l): Worth: New York, 1982;pp 8sL690. (13) Metzler. D. E. "Biochemistry, The Ch.rni"d Reactions of Living Cells"; Acxdemie, New York. 1977:p 658. (14) Speck. J. F. J.BX Chrm. 1949,179,1405. (151 Meister, A. Methods Enrymol. 1970,17A, m. (16) Shspiro, B.:Sfsdtman.E. R. MdhodsEnrymol. 1370.17A. 910. (171 Snoke. J. E.:Ysnari,S.;Bloeh,K. J. Riol. Chem 1953,201,573. (181 Mooz, E.0.: Meistcr, A.Methods Enzymol. 1971,178,483. (19) O r l m k i . M.; Meister, A. Methods En8ymol. 1971,178,495, (20) Lipmann. F. Science 137L, 173,875. (21) Lipmann.F. In "MolecularEvolution,Prcbiol~ieslsndBiological': Roh1fing.D. L.: Opsrin, A. I.. Eds.: Plenum. New York, 1972;pp 261-269. (221 Lipmann. F. I" "From Theoretical Physi- to Biology',: Mami.. M. Ed.; Ksrger, Basel. L911:oo 186.174
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