N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 58

For example, Lys525 is a predominant site of N-homocysteinylation in human
serum albumin [96], while in human fibrinogen α -Lys562, β -Lys344, and

γ -Lys385 are predominant sites for N-homocysteinylation [215].

Studies with human serum albumin show that the rate of protein N-homocystei-
nylation is first order with respect to Hcy-thiolactone and protein concentration.
The pH dependence of pseudo-first-order rate constant of N-homocysteinylation
(Fig. 3.3c ) is consistent with the ionization of Hcy-thiolactone with pK a ¼

6.67
(Reaction 3.3 ) and suggests that the positively charged acid form of Hcy-
thiolactone is more reactive with protein (k ¼ 764 M/h) than the neutral base
form (k ¼ 4 M/h) (Table 3.2 ). The structures of the acid and base forms of Hcy-
thiolactone are illustrated in Reaction 3.3 . The reaction of Hcy-thiolactone with free
lysine exhibits similar pH dependence (Fig. 3.3b ).
Hcy is quantitatively recovered (as Hcy-thiolactone in the presence of a reducing
agent) from N-Hcy proteins only by acid hydrolysis [78, 79]. In fact, this principle is
a basis for quantification of N-linked Hcy content in proteins [297].
Analysis by Edman degradation of a sample of N-homocysteinylated human
serum albumin, containing 3 mol Hcy/mol protein, shows that two major products
are released in the first cycle (as a phenylthiohydantoin, PTH, derivative): PTH-
Hcy and PTH-Asp. This outcome indicates that a free amino group is present on
N-linked Hcy and that the -amino group of N-terminal aspartic acid residue is not

susceptible to N-homocysteinylation; if free amino groups were absent, no PTH-
Hcy and PTH-Asp derivatives would have been recovered [78].
Mass spectrometric analyses have also identified that only

α

ε -amino groups of

internal lysine residues, but not -amino group of the N-terminal amino acid, are

targets for Hcy-thiolactone modification in human serum albumin [96, 212, 213],
hemoglobin [68, 214], cytochrome c [298], fibrinogen [175, 215], and dynein [299].
So far, specific lysine residues susceptible to N-homocysteinylation are known
for several proteins. In addition to the predominant Lys525 residue [96], six other
lysine residues in human serum albumin are susceptible to the modification by Hcy-
thiolactone in vitro: Lys4, Lys12, Lys137, Lys159, Lys205, and Lys212 [212, 213].
Seventeen lysine residues in human fibrinogen susceptible to the modification by
Hcy-thiolactone have been identified [215]: five in α-subunit [Lys562 and Lys52
(major), Lys70, Lys81, and Lys129 (minor)], five in

α

-subunit [Lys344 and Lys396
(major), Lys148, Lys217, and Lys298 (minor)], and seven in

β

-subunit [Lys385,
Lys266, and Lys373 (major), Lys85, Lys95, Lys170, and Lys273 (minor)]. Three of
these residues,

γ

-subunit Lys298, have originally
been found to be susceptible to N-homocysteinylation in vitro by other investigators
[175]. Fourteen sites in hemoglobin susceptible to the modification by Hcy-
thiolactone have been identified [214]: seven in

α -subunit Lys52 and Lys129 and β

α -subunit (Lys7, 11, 16, 56, 90,

99, and 139) and seven in -subunit (Lys8, 17, 59, 61, 66, 82, 95, 120, and 144). In

cytochrome c, 4 lysine residues (Lys8 or -13, Lys86 or -87, Lys99, and Lys100) are
susceptible to N-homocysteinylation in vitro [298].

β