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.
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.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis*; D002250:Carbon Radioisotopes; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D013293:Enterococcus faecalis / Q000201:enzymology / Q000378:metabolism*; D005492:Folic Acid / Q000378:metabolism*; D005561:Formates; D008274:Magnesium / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D012343:RNA, Transfer / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism; D014166:Transferases / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D005561:Formates; D013763:Tetrahydrofolates; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D005492:Folic Acid; D008715:Methionine; D014166:Transferases; D000604:Amino Acyl-tRNA Synthetases; D008274:Magnesium