N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 56

Chapter 5
N-Homocysteinyl-Proteins

5.1 Synthesis In Vitro

During Hcy-thiolactone biosynthesis, a high-energy bond of ATP is conserved in
the thioester bond of Hcy-thiolactone (Reactions 3.7 and 3.8 ). This high-energy
thioester bond is responsible for the chemical reactivity of Hcy-thiolactone toward
amino groups in proteins [73, 78, 139].
When [ 35 S]Hcy-thiolactone is added to human plasma, it disappears with a half-
life of ~1 h at 37 C [78], 25-fold faster than expected from the rate of spontaneous
Hcy-thiolactone hydrolysis [73]. After 3 h most of the radioactivity from [ 35 S]Hcy-
thiolactone is incorporated covalently into protein [73, 78]. Reduction with DTT
releases 30 % of the incorporated [ 35 S] radiolabel as free reduced [ 35 S]Hcy. The
DTT-resistant fraction of the [ 35 S]Hcy-protein adducts is also resistant to
treatments with reagents or enzymes that destroy ester or anhydride bonds, such
as NaOH, hydroxylamine, or rabbit esterase. These findings indicate that protein
amino groups form stable adducts with Hcy-thiolactone while protein hydroxyl and
carboxyl groups are not involved. That protein lysine residues are involved is
supported by the finding that Hcy-thiolactone reacts preferentially with free lysine
forming an N-Hcy-Lys adduct that can be separated from unreacted lysine and Hcy-
thiolactone by thin-layer chromatography [73]. Subsequent NMR studies have
established that the adduct's structure is that of the isopeptide Nε

-Hcy-Lys [72].
In vitro reactions of Hcy-thiolactone with lysine (Fig. 3.3b ) and serum albumin
(Fig. 3.3c ) exhibit similar dependences on pH, which suggests that a similar
mechanism is involved in both reactions. Furthermore, of the two ionic species of
Hcy-thiolactone (Reaction 3.3 ), the positively charged acid form, less abundant at
physiological pH 7.4, is much more reactive than the more abundant neutral base

form, both toward free lysine and protein lysine (Table 3.2 ).
Polyacrylamide gel electrophoretic analyses under reducing conditions show
that in serum incubated with [ 35 S]Hcy-thiolactone, each serum protein becomes 35 S
labeled. Bands corresponding to major serum proteins, such as albumin (67 kDa),
γ

¼

-globulin (50 and 25 kDa), fibrinogen (47, 56, and 67 kDa), transferrin (80 kDa),