This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Samuel, C. E. Articles by Rabinowitz, J. C. Search for Related Content PubMed PubMed Citation Articles by Samuel, C. E. Articles by Rabinowitz, J. C.

Initiation of Protein Synthesis by Folate-Sufficient and Folate-Deficient Streptococcus faecalis R: Partial Purification and Properties of Methionyl-Transfer Ribonucleic Acid Synthetase and Methionyl-Transfer Ribonucleic Acid Formyltransferase

^a Department of Biochemistry, University of California, Berkeley, California 94720

ABSTRACT

The initiation of protein synthesis by Streptococcus faecalis R grown in folate-free culture occurs without N-formylation or N-acylation of methionyl-tRNA_f^Met. Methionyl-tRNA synthetase and methionyl-tRNA formyltransferase were partially purified from S. faecalis grown under normal culture conditions in the presence of folate (plus-folate); the general properties of the enzymes were determined and compared with the properties of the enzymes purified from wild-type cells grown in the absence of folate (minus-folate). S. faecalis methionyl-tRNA synthetase displays optimal activity at pH values between 7.2 and 7.8, requires Mg²⁺, and has an apparent molecular weight of 106,000, as determined by gel filtration, and 127,000, as determined by sucrose density gradient centrifugation. The K_m values of plus-folate methionyl-tRNA synthetase for each of the three substrates in the aminoacylation reaction (L-methionine, adenosine triphosphate, and tRNA) are nearly identical to the respective substrate Michaelis constants of minus-folate methionyl-tRNA synthetase. Furthermore, both plus- and minus-folate S. faecalis methionyl-tRNA synthetases catalyze, at equal rates, the aminoacylation of tRNA_f^Met and tRNA_m^Met isolated from either plus-folate or minus-folate cells. S. faecalis methionyl-tRNA formyltransferase displays optimal activity at pH values near 7.0, is stimulated by Mg²⁺, and has an apparent molecular weight of approximately 29,900 when estimated by sucrose density gradient centrifugation. The K_m value of plus-folate formyltransferase for plus-folate Met-tRNA_f^Met does not differ significantly from that of minus-folate formyltransferase for minus-folate Met-tRNA_f^Met. Both enzymes can utilize either 10-formyltetrahydrofolate or 10-formyltetrahydropteroyltriglutamate as the formyl donor; the Michaelis constant for the monoglutamyl pteroyl coenzyme is slightly less than that of the triglutamyl pteroyl coenzyme for both transformylases. Tetrahydrofolate and uncharged tRNA_f^Met are competitive inhibitors of both plus- and minus-folate S. faecalis formyltransferase; folic acid, pteroic acid, aminopterin, and Met-tRNA_m^Met are not inhibitory. These results indicate that the presence or absence of folic acid in the culture medium of S. faecalis has no apparent effect on either methionyl-tRNA synthetase or methionyl-tRNA formyltransferase, the two enzymes directly involved in the formation of formylmethionyl-tRNA_f^Met. Therefóre, the lack of N-formylation of Met-tRNA_f^Met in minus-folate S. faecalis is due to the absence of the formyl donor, a 10-formyl-tetrahydropteroyl derivative. Although the general properties of S. faecalis methionyl-tRNA synthetase are similar to those of other aminoacyl-tRNA synthetases, S. faecalis methionyl-tRNA formyltransferase differs from other previously described transformylases in certain kinetic parameters.

¹ Present address: Department of Microbiology and Immunology, Duke University Medical Center, Durham, N.C. 27710.

This article has been cited by other articles:

• Urbonavicius, J., Skouloubris, S., Myllykallio, H., Grosjean, H. (2005). Identification of a novel gene encoding a flavin-dependent tRNA:m5U methyltransferase in bacteria--evolutionary implications. Nucleic Acids Res 33: 3955-3964 [Abstract] [Full Text]