Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 33

Reaction 3.6 The formation of aminoacyl adenylate catalyzed by AARS
Reaction 3.7 The aminoacylation of tRNA by AARS
Reaction 3.8 The formation of Hcy-thiolactone catalyzed by MetRS
presence of Hcy and ATP, MetRS catalyzes the formation of homocysteinyl-
adenylate (Hcy-AMP).
That Hcy is a substrate for MetRS was first found in in vitro studies with purified
E. coli and Bacillus stearothermophilus enzymes [232, 233]. However, in contrast
to the cognate methionine which is transferred from Met-AMP to tRNA Met , the
misactivated Hcy is not transferred from Hcy-AMP to tRNA Met [233].
Instead, Hcy-AMP is edited by the conversion to Hcy-thiolactone in an intramo-
lecular reaction in which the side chain thiolate of Hcy displaces the AMP group
from the carboxylate of the misactivated Hcy (Reaction 3.8 ) [210]. Thin-layer
chromatography (TLC) shows that a new compound forms when MetRS is
incubated with Hcy and ATP but is absent in incomplete reaction mixtures missing
any of the three components. This new compound is identified as Hcy-thiolactone.
It separates from Hcy on cellulose or silica gel TLC plates and is visualized under
UV and by staining with ninhydrin. It cochromatographs with an authentic Hcy-
thiolactone standard, absorbs UV light, and gives the same yellow color with
ninhydrin as the standard [210]. Two pages from the author's laboratory notebook
describing the original experiment in which the formation of Hcy-thiolactone
during Hcy editing by MetRS has been discovered are shown in Fig. 3.5 .
In the Hcy editing reaction, 1.03 mol ATP is hydrolyzed per mol of Hcy-
thiolactone formed, which indicates that the cyclization by intramolecular thioester
bond formation, rather than hydrolysis, is a favored reaction of Hcy-AMP [234].
The Hcy editing reaction is not affected by tRNA Met [210].
The Hcy-thiolactone formation reaction corrects an error in amino acid selec-
tion, prevents Hcy from accessing the genetic code for methionine [6, 7], and is
conserved in evolution from bacteria to human beings [64, 93, 197]. Hcy editing,
one of a few error-correcting reactions that can be directly examined in living
organisms, is a textbook paradigm of in vivo editing reactions in the translation of
the genetic code [235, 236].
The involvement of MetRS in the biosynthesis of Hcy-thiolactone in vivo was
first reported in microorganisms, such as E. coli [197] and the yeast Saccharomyces