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

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 Mg 2+ , 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 Mg 2+ , 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.