Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1971 Dec;108(3):1008–1016. doi: 10.1128/jb.108.3.1008-1016.1971

Substrate Specificity of a Mutant Alanyl-Transfer Ribonucleic Acid Synthetase of Escherichia coli

Peter Buckel 1, Werner Lubitz 1, August Böck 1
PMCID: PMC247182  PMID: 4945179

Abstract

The correlation between the in vivo functioning and the in vitro behavior of the thermolabile alanyl-transfer ribonucleic acid (tRNA) synthetase (ARS) of Escherichia coli strain BM113 is presented. As a measure for the ARS activity inside the cell, the amount of acylated tRNAala in vivo was determined. The rapid drop of the per cent tRNAala charged which was observed upon shifting a culture of BM113 to the nonpermissive temperature indicates that in vivo acylation of tRNAala might be the growth-limiting step at high temperature. Since neither growth nor the in vivo charging level of tRNAala was affected by the addition of high l-alanine concentrations to the medium, one may infer that impaired functioning of the mutant enzyme at 40 C seems not to be due to reduced affinity of the enzyme for the amino acid. Separation of bulk tRNA of E. coli and of yeast on benzoylated diethylaminoethyl cellulose and charging of the fractions of the column by wild-type and mutant ARS reveal that only those tRNA species aminoacylated by the wild-type enzyme are also charged by the mutant ARS. Determination of the Km values of wild-type and mutant ARS for the three isoaccepting tRNAala species of E. coli shows a ca. 10-fold increase of the apparent Km values of the mutant enzyme for all three species. Thus, the mutation proportionally reduces the apparent affinity for tRNAala without causing any detectable recognition errors. Investigation of heat inactivation kinetics of wild-type and mutant ARS without and in the presence of substrates provides further evidence that only the transfer site of the ARS is altered by the mutation. Moreover, whereas both enzymes possess the same pH optimum of the relative maximal velocity, their pH dependence of the Km values for tRNA is different. The Km of the wild-type enzyme decreases at pH values below 7.0 and that of the mutant enzyme shows the inverse tendency; this again indicates an alteration of the tRNA binding site.

Full text

PDF
1008

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexander R. R., Calvo J. M., Freundlich M. Mutants of Salmonella typhimurium with an altered leucyl-transfer ribonucleic acid synthetase. J Bacteriol. 1971 Apr;106(1):213–220. doi: 10.1128/jb.106.1.213-220.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BARNETT W. E., JACOBSON K. B. EVIDENCE FOR DEGENERACY AND AMBIGUITY IN INTERSPECIES AMINOACYL-SRNA FORMATION. Proc Natl Acad Sci U S A. 1964 Apr;51:642–647. doi: 10.1073/pnas.51.4.642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BURTON K. The stabilization of D-amino acid oxidase by flavin-adenine dinucleotide, substrates and competitive inhibitors. Biochem J. 1951 Apr;48(4):458–467. doi: 10.1042/bj0480458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Böck A. Mutation affecting the charging reaction of alanyl-tRNA synthetase from Escherichia coli K 10. Arch Mikrobiol. 1969 Oct;68(2):165–178. doi: 10.1007/BF00413875. [DOI] [PubMed] [Google Scholar]
  5. Böck A., Neidhardt F. C. Location of the structural gene for glycyl ribonucleic acid synthetase by means of a strain of Escherichia coli possessing an unusual enzyme. Z Vererbungsl. 1966;98(3):187–192. doi: 10.1007/BF00888946. [DOI] [PubMed] [Google Scholar]
  6. Chuang H. Y., Atherly A. G., Bell F. E. Protection of the proline-and valine-activating enzymes by their amino acid substrates against thermal inactivation. Biochem Biophys Res Commun. 1967 Sep 27;28(6):1013–1018. doi: 10.1016/0006-291x(67)90082-4. [DOI] [PubMed] [Google Scholar]
  7. Doolittle W. F., Yanofsky C. Mutants of Escherichia coli with an altered tryptophanyl-transfer ribonucleic acid synthetase. J Bacteriol. 1968 Apr;95(4):1283–1294. doi: 10.1128/jb.95.4.1283-1294.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. FANGMAN W. L., NEIDHARDT F. C. DEMONSTRATION OF AN ALTERED AMINOACYL RIBONUCLEIC ACID SYNTHETASE IN A MUTANT OF ESCHERICHIA COLI. J Biol Chem. 1964 Jun;239:1839–1843. [PubMed] [Google Scholar]
  9. FRAENKEL D. G., NEIDHARDT F. C. Use of chloramphenicol to study control of RNA synthesis in bacteria. Biochim Biophys Acta. 1961 Oct 14;53:96–110. doi: 10.1016/0006-3002(61)90797-1. [DOI] [PubMed] [Google Scholar]
  10. Folk W. R., Berg P. Characterization of altered forms of glycyl transfer ribonucleic acid synthetase and the effects of such alterations on aminoacyl transfer ribonucleic acid synthesis in vivo. J Bacteriol. 1970 Apr;102(1):204–212. doi: 10.1128/jb.102.1.204-212.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gillam I., Millward S., Blew D., von Tigerstrom M., Wimmer E., Tener G. M. The separation of soluble ribonucleic acids on benzoylated diethylaminoethylcellulose. Biochemistry. 1967 Oct;6(10):3043–3056. doi: 10.1021/bi00862a011. [DOI] [PubMed] [Google Scholar]
  12. Hirshfield I. N., Horn P. C., Hopwood D. A., Maas W. K., DeDeken R. Studies on the mechanism of repression of arginine biosynthesis in Escherichia coli. 3. Repression of enzymes of arginine biosynthesis in arginyl-tRNA synthetase mutants. J Mol Biol. 1968 Jul 14;35(1):83–93. doi: 10.1016/s0022-2836(68)80038-5. [DOI] [PubMed] [Google Scholar]
  13. Iaccarino M., Berg P. Isoleucine auxotrophy as a consequence of a mutationally altered isoleucyl-transfer ribonucleic acid synthetase. J Bacteriol. 1971 Feb;105(2):527–537. doi: 10.1128/jb.105.2.527-537.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. James H. L., Morrison J. C., Shiflet R. N., Trass T. C., Whybrew W. D., Bucovaz E. T. Interaction of homologous transfer RNA with yeast aminoacyl-RNA synthetases. Biochem Biophys Res Commun. 1968 Nov 25;33(4):574–583. doi: 10.1016/0006-291x(68)90334-3. [DOI] [PubMed] [Google Scholar]
  15. Kelmers A. D., Novelli G. D., Stulberg M. P. Separation of transfer ribonucleic acids by reverse phase chromatography. J Biol Chem. 1965 Oct;240(10):3979–3983. [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. Lazar M., Yaniv M., Gros F. Sur les propriétés d'une alanyl-t-RNA synthétase modifiée dans une souche d'Escherichia coli à croissance thermosensible. C R Acad Sci Hebd Seances Acad Sci D. 1968 Jan 29;266(5):531–534. [PubMed] [Google Scholar]
  18. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  19. Mitra S. K., Chakraburtty K., Mehler A. H. Binding of transfer RNA and arginine to the arginine transfer RNA synthetase of Escherichia coli. J Mol Biol. 1970 Apr 14;49(1):139–156. doi: 10.1016/0022-2836(70)90382-7. [DOI] [PubMed] [Google Scholar]
  20. Nass G. Regulation of histidine biosynthetic enzymes in a mutant of Escherichia coli with an altered histidyl-tRNA synthetase. Mol Gen Genet. 1967;100(2):216–224. doi: 10.1007/BF00333608. [DOI] [PubMed] [Google Scholar]
  21. Neidhardt F. C. Roles of amino acid activating enzymes in cellular physiology. Bacteriol Rev. 1966 Dec;30(4):701–719. doi: 10.1128/br.30.4.701-719.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schlesinger S., Nester E. W. Mutants of Escherichia coli with an altered tyrosyl-transfer ribonucleic acid synthetase. J Bacteriol. 1969 Oct;100(1):167–175. doi: 10.1128/jb.100.1.167-175.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Szentirmai A., Szentirmai M., Umbarger H. E. Isoleucine and valine metabolism of Escherichia coli. XV. Biochemical properties of mutants resistant to thiaisoleucine. J Bacteriol. 1968 May;95(5):1672–1679. doi: 10.1128/jb.95.5.1672-1679.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Taglang R., Waller J. P., Befort N., Fasiolo F. Amino-acylation du tRNA-1-Val de Escherichia coli par la phénylalanyl-tRNA synthétase de levure. Eur J Biochem. 1970 Feb;12(3):550–557. doi: 10.1111/j.1432-1033.1970.tb00886.x. [DOI] [PubMed] [Google Scholar]
  25. Weiss J. F., Kelmers A. D. A new chromatographic system for increased resolution of transfer ribonucleic acids. Biochemistry. 1967 Aug;6(8):2507–2513. doi: 10.1021/bi00860a030. [DOI] [PubMed] [Google Scholar]
  26. Yaniv M., Gros F. Studies on valyl-tRNA synthetase and tRNA from Escherichia coli. 3. Valyl-tRNA synthetases from thermosensitive mutants of Escherichia coli. J Mol Biol. 1969 Aug 28;44(1):31–45. doi: 10.1016/0022-2836(69)90403-3. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES