STUDIES ON SOME INTERMEDIATE REACTIONS IN BACTERIAL PROTEIN SYNTHESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By ROBERT WILLIAM BERNLOHR, B. S The Ohio State University 1958 Approved by Department of Agricultural Biochemistry ACKNOWLEDGEMENTS I wish to express ray appreciation to Dr. George C. Webster for his advice and helpful suggestions during the course of this research program. The help of Dr. J. E, Varner and the other faculty members is also greatly acknowledged. I am indebted to the Department of Agricultural Biochemistry for the financial assistance and facilities made available to me for the conduction of this project. Also, the financial assistance of the Charles P. Kettering Foundation and the Public Health Service is acknowledged. The encouragement, sacrifices, and help received from my wife during the past three years and in typing this manuscript are deeply appreciated. ii CONTENTS Page Introduction 1 Experimental Procedures 11+ Results and Discussion I. Whole Cell Studies on Amino Acid Incorporation 26 II* Cell-free Incorporating Systems i}2 Ill* Amino Acid Activations I4.7 IV. Intermediates in the Biosynthesis of Protein £2 Summary 68 Bibliography 72 Autobiography 77 iii LIST OF TABLES Table Page 1. Glycine-C^+ Incorporation Into Different Tissues 27 2. Changes in RNA, DNA, and Protein 28 3. Effect of RNAase and DNAase 31+ 1+. Effect of Chloramphenicol 37 $- Cell-free Amino Acid Incorporation 1+5 6. Oxygen Transfer in Amino Acid Activation 1+9 7. Chromatography of Amino Acid Hydroxamates 55 8. Distribution of AA-X 57 9. Sensitivity of AA-X to pH and Dialysis 58 10. Kinetic Uptake of Glycine-C1^ 67 iv LIST OF FIGURES Figure Page 1. Glycine-C1^ Incorporation into RNA, DNA, and Protein 30 2. Effect of Chloramphenicol 33 3. Inhibition of RNA, DNA, and Protein Synthesis 35 h- Distribution of Glyclne-C^ in Centrifugal Fractions 39 5. Electrophoretic and Ultracentrifugal Analysis of Particulate Fractions kk 6* Chromatography of Amino Acid Hydroxamates 56 7. Spectra of the Dialyzed and Treated Supernatant Solution 60 8. Specific Activity of Components Separated by Paper Electrophoresis 62 9. Total Activity of Components Separated by Paper Electrophoresis 63 v INTRODUCTION The elucidation of the enzymatic mechanism of protein synthesis is currently a very pressing problem, for it remains as the last major void in biochemical knowledge. Daily, hundreds of people are admitted to hospitals with conditions which Involve accelerated cell growth, such as cancer. In addition, a large number of physiological abnormalities can be traced to the occurrence of unnatural protein molecules. These two examples, to which many more could be added, indicate that the sensitive biosynthetic system responsible for the production of protein molecules is unusually prone to malfunction. Also the genetic apparatus, present in all cells, appears to be at least indirectly involved in organizing this process. It Is evident, therefore, that a knowledge of the stepwise biosynthesis of proteins could conceivably lead to an understanding of the cause of many pathological conditions. It is of considerable importance, then, to have a well-defined hypothesis at the level of reaction mechanisms in terms of chemical formulas. In considering the possible mechanism for protein biosynthesis, several problems are encountered which are unique for this type of molecule. The first is the 1 formation of peptide bonds. Secondly, proteins are constructed from a number of different amino acids which are apparently arranged in a specific sequence and main­ tained from generation to generation. Thus differing from polysaccharides and lipids, they seem to owe this specificity to direct genetic control, and therefore, a regulating mechanism is required. The initial activation of the constituent amino acids and model systems for peptide bond formation have received considerable attention. Prom previous studies of model systems for peptide bond formation, Lipmann (191 +5 ) discovered that during the acylation of aromatic amines, adenosine triphosphate (ATP) was utilized for carboxyl activation. Also in glutathione synthesis £Bloch (19219 )} t a similar reaction was observed in which ATP is split to form adenosine diphosphate (ADP) and inorganic ortho­ phosphate. However, a study of the synthesis of acetyl-CoA by Lipmann £t al. (1952) provided an alternative mechanism for the activation of carboxyl compounds. When the enzymatic reaction involving acetate, ATP, and coenzyrae A is observed, the products are acetyl-CoA, adenosine monophosphate (AMP), and inorganic pyrophosphate (PPi). Furthermore, this reaction, representing a new type of cleavage of ATP, is freely reversible and allows the exchange of radioactive pyrophosphate into ATP. We are then presented with two schemes in which the phosphate bond energy of ATP could be used to drive the energy requiring synthesis of proteins* The two possibilities for the mechanism of amino acid activation and subsequent condensation are --- n AA + n ATP ^ (AA)n + n ADP + n Pi or n AA + n ATP -r (AA)n + n AMP + n PPi. The activation of amino acids was demonstrated when extracts of animal, plant, and bacterial systems were shown to catalyze an amino acid dependent exchange between ATP and inorganic pyrophosphate by Hoagland (1955) Hoagland, Keller and Zamecnik (1955); Webster (1957); Holley (1957); and Novelli and DeMoss (1958)* In Hoagland’s original work, hydroxylamine was also used as a trapping agent for the activated amino acid, indicating that the carboxyl group of the amino acid is the site of activation. From this work and also from that of Berg (1957) the occurrence of an enzyme-bound amino acyl-AMP compound has been postulated as the activated amino acid. After the amino acids have been activated, it is assumed that there is an enzyme present which will catalyze the union of a series of these building blocks by peptide formation. However, a regulatory mechanism must be applied to ensure the formation of specific completed proteins. Otherwise, as Steinberg and Anfinsen (1952) k- believed, peptide intermediates are possible, Simpson and Velick (195U-) and Askonas et al. (195U) have since shown that the intracellular biosynthesis of peptide intermediates during the formation of proteins is not very likely. Many formulations for the biosynthesis of protein have been proposed. One mechanism implies that at each step, contact between the appropriate catalyst and the molecule being farmed is limited to relatively small portions of the molecular structure of either. Conse­ quently, each of the many catalysts can have but limited information of what has gone before. This polyenzyme theory QSpiegelman (1957)3 runs into such inherent complications and difficulties that it must be abandoned in favor of a more controllable mechanism. The familiar template theory, first stated by Breinl and Haurowitz (1930) and generalized by Haurowitz (1950), can account for the exact replication of specific protein molecules, and embodies most of the existing postulated synthesis mechanisms. This theory has as its basis the duplication of protein molecules on the surface of another protein which is fixed in an expanded state by conjugation with a nucleic acid molecule. Approaching the problem in more specific chemical terms, Bounce (1952) suggested that the activating enzyme, 5 bound to nucleic acid, is the tenplate itself. In his opinion, ATP contributes the necessary energy by means of a phosphotransferase that transfers its terminal phosphate to the phosphate of nucleic acid; the net result is the transfer of a pyrophosphate linkage from ATP to nucleic acid. The amino groups of amino acids then react to form amino-phosphate compounds on the nucleic acid, and for each amino group so combined the phosphate that came originally from the ATP is displaced and appears as inorganic phosphate. Another enzyme links the free carboxyl group of the adjacent amino acid to the phosphate-bound amino group to form a peptide linkage. Lipmann (19Sk) postulated that a pyrophosphoryl moiety from ATP is transfer:-ed to the nucleic acid template. It is then displaced by the carboxyl groups of amino acids and upon the influence of an additional enzyme, peptide bond formation between adjacent amino acids takes place, allowing the completed protein to leave the template structure. By 195b, Borsook had incorporated the newly acquired knowledge concerning amino acid activation into his hypothesis for protein biosynthesis. He feels that after amino acids have been activated, they are attached to the phosphate of nucleic acids by their carboxyl groups. Then 6 another enzyme forms the peptide bonds, thereby removing the amino acids involved from the nucleic acid template. Recently, Hoagland e_t al. (1957a) and Holley (1957) have postulated the occurrence of anadditional cofactor necessary for protein synthesis. Novelli and DeMoss (1 9 5 8) have subsequently utilized this information in formulating a hypothesis for the biosynthesis of protein. They feel that the enzyme-bound activated amino acid is transferred to a factor "X" which can be considered as a transport cofactor. The amino acid moiety of this complex is then donated to a template, and polymerization occurs in the manner described by Borsook (195b). At the present time, the limiting factor in the elucidation of this mechanism appears to be a lack of information concerning the chemical nature of the template.