Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 42

Table 3.8 Relative binding, editing, and tRNA aminoacylation under steady-state conditions by
E. coli MetRS (Compiled from [65, 75, 210])
Amino acid Binding Rate of editing Rate of tRNA aminoacylation

Methionine 100 1 100

S-nitroso-Hcy 7.63 1 1.2

<

0.0001 a Homocysteine 1.13 60

<

a Hcy is not transferred to tRNA

Table 3.9 Translational and posttranslational incorporation of Hcy into protein in cultured human
endothelial cells (Data from [76])

Translational Posttranslational

[ 35 S]Hcy-protein (%) [ 35 S]Met-protein (%) N-[ 35 S]Hcy-protein (%) Labeling conditions

[ 35 S]Hcy, 80 μM 57 19 24

[ 35 S]Hcy, 40 μM 47 20 33

[ 35 S]Hcy, 10 μM

37 25 38

[ 35 S]Hcy, 10 μM + folate,
10 μM

< 1 > 98 < 1

[ 35 S]Hcy, 10 μM + Met,
20

12 76 12

μ M

[ 35 S]Hcy, 10

M + HDL,
1mgmL 1

μ 68 25 7

εN-[ 35 S]Hcy-
Lys-protein

Control, 4 0 96

< >

Human umbilical vein endothelial cells (HUVECs) are maintained on Met-free M199 culture
medium, supplemented with dialyzed 15 % fetal bovine serum, bovine endothelial cell growth
factor, heparin, and indicated concentration of [ 35 S]Hcy (50 Ci mL 1 ), in the absence and

presence of exogenous folate, HDL or Met. [ 35 S]Met-protein and protein-[ 35 S]Hcy (total
incorporation of Hcy into protein) are determined by acid hydrolysis of extracellular proteins
followed by thin-layer chromatography. [ 35 S]Hcy-protein (translationally incorporated Hcy) and
N-[ 35 S]Hcy-protein (posttranslationally incorporated Hcy) are distinguished the susceptibility to
Edman degradation: N-[ 35 S]Hcy-protein is sensitive to Edman degradation, while [ 35 S]Hcy-pro-
tein and [ 35 S]Met-protein are resistant. Relative distribution (%) of the [ 35 S]radioactivity among
indicated chemical species observed under each experimental condition is shown

μ

Hcy-containing Dhfr, globin, and luciferase are indistinguishable from the
native Met-containing proteins on SDS-PAGE gels [75]. This shows that Hcy
incorporation into protein in place of Met does not lead to breakage of peptide
bonds and that Hcy is compatible with protein structure. Other studies have shown
that Hcy can be incorporated in place of cysteine by chemical synthesis into
peptides such as the hormone oxytocin [247] and the isopenicillin precursor δ - L -

α -aminoadipoyl- L -homocysteinyl- D -valine (AhCV) [248]. Resulting Hcy-

containing peptides are chemically stable. While Hcy-oxytocin is devoid of
biological activity of normal oxytocin [247], AhCV is a substrate for isopenicillin
synthase, which oxidizes AhCV to a -lactam

arising from the oxidation of the natural cysteine-containing precursor [248].
Translational incorporation of Hcy into protein occurs in cultured human vascu-
lar endothelial cells (Table 3.9 ) [76, 83] that are known to produce nitric oxide and

δ -lactam, a higher homolog of the γ