Amino acid-activating enzymes

As we have noted, the outcome of a virus infection is the synthesis of viral nucleic acid and viral protein coats. In effect, the virus takes over the biosynthetic machinery of the host and uses it for its own synthesis. A few enzymes needed for virus replication may be present in the virus particle and may be introduced into the cell during the infection process, but the host supplies everything else energy-generating system, ribosomes, amino-acid activating enzymes, transfer RNA (with a few exceptions), and all soluble factors. The virus genome codes for all new proteins. Such proteins would include the coat protein subunits (of which there are generally more than one kind) plus any new virus-specific enzymes. 

Fig. 5.1 Simplified model representation of the activation of an amino acid (ASY) at an amino acid-activating enzyme (i.e., an amino acid-specific aminoacyl-tRNA synthetase)...

Jhe synthesis of proteins, as characterized by the in vitro incorporation of amino acids into the protein component of cytoplasmic ribonu-cleoprotein, is known to require the nonparticulate portion of the cytoplasm, ATP (adenosine triphosphate) and GTP (guanosine triphosphate) (15, 23). The initial reactions involve the carboxyl activation of amino acids in the presence of amino acid-activating enzymes (aminoacyl sRNA synthetases) and ATP, to form enzyme-bound aminoacyl adenylates and the enzymatic transfer of the aminoacyl moiety from aminoacyl adenylates to soluble ribonucleic acid (sRNA) which results in the formation of specific RNA-amino acid complexes—see, for example, reviews by Hoagland (12) and Berg (1). The subsequent steps in pro-... 

Experimentally, C14-aminoacyl sRNA was incubated with rat liver microsomes or ribosomes, GTP, various fractions obtained from the nonparticulate portion of rat liver homogenates, and buffered salt-sucrose medium in a total volume of approximately 2 ml. (6-10). The C14-aminoacyl sRNA was prepared by the phenol-extraction procedure from the pH 5 amino acid-activating enzymes, fraction of rat liver after incubation with C14-L-amino acids (9, 13). C14-leucyl sRNA (approximately 1000 c.p.m.), having a specific radioactivity of approximately 55,000 c.p.m. per mg. of RNA, and containing a complement of endogenous, unlabeled, bound amino acids, was used in most of these studies. The microsomes were sedimented from the post-mitochondrial supernatant at 104,000 x g (10) and the ribosomes were prepared from them by extraction with deoxycholate (16). 

Previous studies by Hoagland et al. (13), Zamecnik et al. (24), and in this laboratory (9, 10) demonstrated that the transfer of amino acid from isolated sRNA-amino acid to microsomes required GTP, ATP, an ATP-generating system, and a soluble portion of the cell. Most of the aminoacyl-transferring activity present in the homogenate supernatant was recovered in the pH 5 Supernatant obtained after precipitation of the amino acid-activating enzymes at pH 5. A protein fraction, 500- to... 

P4. Pennington, R. J., Amino acid-activating enzymes in muscle. Biochem. J. TJ, 205-208 (I960). 

Mehler, A.H. Induced activation of amino acid activating enzymes by amino acids and tRNA. Prog. Nucleic Acid Res. Mol. Biol. 10,1-22(1970)... 

Neidhardt, F.C. Roles of amino acid activating enzymes in cellular physiology. Bact. Rev. 30, 701-719 (1966)... 

Amino acid activating enzymes, 24, 26 Amino acid chloromethyl ketones, 200 purification of, 611 synthesis of, 609, 610 Amino acid oxidase, 37 Aminoacyl-tRNA, 103, 104, 624, 626, 629-631, 707... 

Eigneg E.A. and Loftfield R.B. (1974) Kinetic techniques for the investigation of amino acid tRNA ligases (aminoacyl-tRNA synthetases, amino acid activating enzymes). Methods EnzymoL, 29, 601-619. 

Preiss, j., Berg, P., Ofengand, E. J., Bergmann, F. H. and Dieckmann, M. (1959) The chemical nature of the RNA-amino acid compound formed by amino acid-activating enzymes. Proc. Nat. Acad. Sci. 45, 319. 

There are two commonly used assay procedures for investigating amino acid activating enzymes. These are (a) the hydroxamate assay 97, 99, 100, 103, 104) where an extract is incubated in the presence of high concentrations (1-2 M) of hydroxylamine, and the ATP-and amino acid-dependent formation of hydroxamate is measured the latter is best determined by the method of Schweet et al. 105) , and (5) the pyrophosphate exchange assay 97, 99, 100), in which an amino acid-dependent exchange of labeled pyrophosphate with the two terminal phosphates of ATP is measured. 

Within the cell, the bulk of the amino acid activating enzymes occurs... 

From the survey presented it can be seen that the amino acid activating enzymes are indeed ubiquitous. As a matter of fact there is not a single case where these enzymes have been looked for and have not been found. Because of this, their presence is nowadays rather taken for granted and often people no longer bother to test for them. 

Some amino acid activating enzymes have been prepared in highly purified form. These are listed in Table IV they are all specific for a single amino acid, but may be contaminated with 10-50 % of other protein material. 

Fio. 6. Schematic representation of the active site on an amino acid activating enzyme. [Taken from Raacke (t0). ... 

The soluble fraction, or the pH 5 enzymes, contain amino acid activating enzymes, transfer RNA, the enzymes responsible for the terminal addition of nucleotides to the sRNA (and hence for conditioning the latter for accepting amino acids from the activating enzymes) and, probably, enzymes responsible for the transfer of the activated amino acid from the sRNA to the RNP particles. In addition, this fraction contains large amounts of unidentified protein material. 

Let us consider the first question. Since it has been shown that amino acid-RNA compounds can be formed by highly purified amino acid activating enzymes, and the properties of this reaction and of the reaction product correspond closely to those observed in crude systems or in vivo, it seems to be established beyond reasonable doubt that the mechanism of this reaction is as outlined in Section III, B, 3, d. Furthermore, the demonstration that the amino acid bound to transfer RNA can be transferred to the microsomes and be bound there in the interior of a peptide chain, seems to show that the RNA-amino acid compound can serve as a donor of amino acid for the incorporation reaction however, since not only GTP, but also ATP and soluble fraction (which mi t contribute a large number of other factors besides the transferring enzyme) are required for the transfer, the role of sRNA-amino acid is less clear-cut than it might be. A reversal of the reaction back to the adenylates, however, and incorporation by some other route seems to be excluded by the fact that even a hundredfold excess of nonisotopic amino acid does not interfere with the efficiency of the transfer, which under the right conditions, approaches 100% (14S). The evidence to date, then, indicates that the adenylate-sRNA pathway is a pathway of amino acid incorporation in the microsomal system of mammalian origin. 

Fig. 15. Over-all scbematio representation of the reactions involved in the adenylate-sRNA pathway of amino acid incorporation. Ei represents the amino acid activating enzymes E, the transferring enzymes and Es and E4, the enzymes responsible for the terminal incorporation into sRNA of ATP and CTP, respectively.

The previously observed trend of studying certain isolated components of the amino acid incorporatir stem without keeping in mind the interaction of these components in the whole system has continued, so that by far the largest number of publications in the field of protein syntheras are not really concerned with the formation of protein, or even with the incorporation of amino acids into protein, but with the study of amino acid activating enzymes, sRNA, and of course, ribonucleoprotein particles. 

---