Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 36

Fig. 3.6 A relationship
between the cellular levels of
MetRS and Hcy-thiolactone
synthesis in E. coli cells.
Different cellular MetRS
levels are produced by
manipulating the expression
of the MetRS gene cloned in a
plasmid under the control of
β -galactosidase promoter

(Reproduced from [197])

Similarly, treatments with the antifolate drug aminopterin (which prevents
remethylation of Hcy to Met by Met synthase) cause hyperhomocysteinemia
and increase Hcy-thiolactone levels in cultured human cells and in the plant
Lupinus luteus [63].
Experiments with mouse renal adenocarcinoma cells [138], human breast
cancer cells [73], and oncogenically transformed human endothelial cells [64]
show that oncogenic transformation increases Hcy levels and leads to increased
Hcy-thiolactone biosynthesis [237].
(e) Supplementation of growth media with methionine, the natural substrate of
MetRS, competes Hcy out of the MetRS active site and completely abolishes
the biosynthesis of Hcy-thiolactone in E. coli [197] and yeast [63]. Supplemen-
tation with other amino acids does not affect Hcy-thiolactone biosynthesis.
Similar inhibition by methionine of Hcy-thiolactone biosynthesis catalyzed
by MetRS has been recapitulated in vitro [210]. Supplementation with Met
inhibits Hcy-thiolactone biosynthesis also in human endothelial cells [74].

Taken together, these findings provide genetic evidence that MetRS is involved
in Hcy-thiolactone biosynthesis, establish a substrate-product relationship between
Hcy and Hcy-thiolactone in ex vivo cultured cells and in vivo in intact organisms,
suggest that Hcy editing occurs at the active site (whose major role is providing
Met-tRNA for protein biosynthesis), and show that Hcy editing is universally
conserved from the bacteria to humans and is greatly enhanced under pathological
conditions in humans and experimental animals.

3.4.2 Molecular Mechanism

3.4.2.1 The Synthetic/Editing Active Site of Methionyl-tRNA Synthetase

Although studied in several biological systems, molecular mechanism of Hcy
editing is best understood for E. coli MetRS. The Hcy editing reaction occurs in