Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 41

fundamentally different peptide-synthesizing systems have a common evolutionary
origin. This suggestion is further supported by findings showing that several
bacterial species express SerRS homologs that aminoacylate phosphopantetheine
prosthetic group of carrier protein with glycine or alanine but do not aminoacylate
tRNA [246]. These SerRS homologs and the thiol-aminoacylating ability of
aminoacyl-tRNA synthetases may represent molecular fossils linking coded ribo-
somal protein synthesis with noncoded nonribosomal peptide synthesis [199].

3.4.2.3 S-NO-Hcy Is Transferred to tRNA and Participates in Protein

Biosynthesis

In the model of the synthetic/editing site of MetRS [240], the side chain of the
cognate substrate methionine is bound exclusively in the specificity subsite
(Fig. 3.8 , top panel), while the side chain of the non-cognate substrate Hcy
partitions between the specificity subsite and the thiol-binding subsite of MetRS
(Fig. 3.8 , middle panel). This is due to 88-fold weaker interaction of Hcy, relative to
methionine, with the specificity subsite (Table 3.8 ). Thus, by enhancing the binding
of the side chain of Hcy in the specificity subsite, one could prevent editing and
allow Hcy transfer to tRNA Met . This, in fact, has been achieved by exploiting
nitrosothiol chemistry in vitro and in vivo in E. coli [75] and can occur naturally
in nitric oxide (NO)-producing cells, such as human endothelial cells [76].
Hcy is quantitatively converted to S-NO-Hcy using equimolar amounts of
NaNO 2 in 0.1 M HCl [188]. It is found that S-NO-Hcy is a better substrate for
MetRS than Hcy in the aminoacyl adenylate formation reaction, mostly due to 14.3-
fold lower K m value [75]. The k cat /K m value for S-NO-Hcy is 6.7-fold higher than
for Hcy, indicating stronger binding of S-NO-Hcy to the specificity subsite
(Table 3.8 ). This stronger binding to the specificity subsite essentially prevents
binding to the editing subsite. As a result, S-NO-Hcy is not edited, but, instead, is
transferred to tRNA Met forming S-NO-Hcy-tRNA Met (Fig. 3.8 , bottom panel)ata
significant rate (Table 3.8 ) [75].
S-NO-Hcy-tRNA Met is as stable as Met-tRNA Met —both are deacylated with a
half-life of 26 min. In contrast, Hcy-tRNA Met (prepared by de-nitrosylation of
S-NO-Hcy-tRNA Met ) is the least stable aminoacyl-tRNA known (deacylation
half-life of 15 s). Hcy-thiolactone and free tRNA Met are the products of deacylation
of Hcy-tRNA Met [75].
S-NO-Hcy-tRNA Met , similar to Met-tRNA Met , is a substrate for ribosomal pro-
tein biosynthesis [75]. For example, E. coli cells unable to metabolize Hcy to Met
due to the inactivation of the metE gene utilize S-NO-Hcy for protein biosynthesis.
When cultures of E. coli metE mutant cells expressing mouse dihydrofolate reduc-
tase (Dhfr) protein are supplemented with S-NO-Hcy, the synthesized Dhfr
protein is found to contain Hcy. Globin and luciferase produced in an in vitro
mRNA-programmed rabbit reticulocyte protein synthesis system supplemented
with S-NO-Hcy-tRNA Met contain Hcy at positions normally occupied by methio-
nine [75].