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
Authors: Samuel Charles E. , Rabinowitz Jesse C. 
Published: January 2020

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.