Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 40

Table 3.7 Comparison of active site residues of MetRS from different organisms (Adapted from
[241])

Residue number
12 Organism 13 14 257 259 260 297 301

Homo sapiens Ala Leu Pro Thr Gly Tyr Asn His

Saccharomyces cerevisiae Ala Leu Pro Thr Gly Tyr Asn His

Escherichia coli Ala Leu Pro Pro Gly Tyr Ile His

Pyrococcus furiosus Ala Leu Pro Pro Gly Tyr Asn His

Methanocaldococcus jannaschii Ala Leu Ala Pro Gly Tyr Ile His

Thermus thermophilus Pro Ile Tyr Leu Asn Tyr Ile His

Mycobacterium smegmatis Pro Ile Ala Leu Asn Tyr Ile His

The non-cognate substrate Hcy, missing the methyl group of methionine, cannot
interact with the specificity subsite as effectively as the cognate methionine does.
This allows the side chain of Hcy to move to the thiol-binding subsite, which
promotes the synthesis of the thioester bond during editing (Fig. 3.8 , middle panel).
Mutations of Tyr15 and Trp305 affect the ability of MetRS to discriminate between
Hcy and methionine [238]. The active site residue Asp52, which forms a hydrogen
bond with the

-amino group of the substrate methionine, deduced from the crystal
structure of the MetRS•Met complex [239], is involved in the catalysis of both
synthetic and editing reactions but does not contribute to substrate specificity of the
enzyme. The substitution Asp52Ala inactivates both the synthetic and editing
functions of MetRS [75, 238, 240].

α

3.4.2.2 The Thiol-Binding Subsite of Methionyl-tRNA Synthetase

The thiol-binding subsite is responsible for the ability of MetRS to catalyze the
thioester bond formation between a thiol and the cognate methionine (Fig. 3.8 ,
middle panel). The intermolecular thioester bond formation reaction mimics the
intramolecular thioester bond formation reaction during Hcy editing and is dubbed
editing in trans [243]. With CoA-SH or cysteine as a thiol substrate, MetRS
catalyzes the formation of Met-S-CoA thioesters [199] and Met-Cys dipeptides
[240], respectively, while the reaction with pantetheine generates Met-S-pantetheine
thioester. The formation of Met-Cys dipeptide proceeds via a Met-S-Cys thioester
intermediate, which spontaneously rearranges to the Met-Cys dipeptide. The forma-
tion of Met-Cys dipeptide as a result of editing in trans is as fast as the formation of
Hcy-thiolactone during Hcy editing.
Although MetRS (as well as IleRS, ValRS, and LysRS) uses the thiol-binding
site for Hcy editing, such site also appears to be present in aminoacyl-tRNA
synthetases that do not edit Hcy but catalyze the aminoacylation of thiols, such as
ArgRS, AspRS, and SerRS [199, 234, 244, 245]. The thiol aminoacylation reactions
are analogous to pantetheine thiol aminoacylation by the multienzyme complexes
responsible for nonribosomal peptide synthesis. This analogy suggests that the two