N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 75

Such chemical alterations affect structure and biological function of Hcy-
thiolactone-modified proteins. For example, early in vitro studies have shown that
N-homocysteinylation of trypsin and methionyl-tRNA synthetase causes progres-
sive loss of their enzymatic activity with increasing degree of N-homocystei-
nylation [78]. N-Hcy-proteins are prone to oxidative damage [96, 311] and
aggregation [78, 96, 171] and are cytotoxic [170, 171] and immunogenic [134,
135, 172]. Subsequent studies have shown that in vitro N-homocysteinylation
inactivates paraoxonase 1 activity in human HDL [320] and RNase activity of
human serum albumin [321]. Although studied with several proteins, the structural
and functional consequences of N-homocysteinylation are best understood for
albumin and fibrinogen, the known targets for the modification by Hcy-thiolactone
in the human body.
N-Hcy-proteins are novel examples of modified proteins that expand the known
repertoire of nonenzymatic protein modifications [322] by other metabolites, such
as glucose, products of lipid peroxidation, or certain drugs, such as penicillin or
aspirin [78]. These protein modification reactions share two common aspects: (i)
each involves protein lysine residues as sites of modifications and (ii) are linked to
human pathology, such as cardiovascular disease, Alzheimer's disease, diabetes,
and drug allergy or intolerance [78].

5.4.1 N -Homocysteinylation and Redox Function

5.4.1.1 N -Hcy-Albumin

Human serum albumin is the most abundant multifunctional plasma protein present
at a mean concentration of 0.63 mM. Albumin, a globular protein of 66.5 kDa
molecular weight, is composed of 585 amino acid residues, including 56 lysine
residues and 35 cysteine residues, 34 of which form 17 disulfide bonds and one,
Cys-34, has a free thiol [323]. It is synthesized in the liver and has a half-life of 19

days in the circulation.
The first detailed studies of structural and functional alterations in a protein
caused by N-homocysteinylation have been carried out with human serum albumin
[96], in which Lys-525 is a predominant site for N-homocysteinylation both in vitro
and in vivo in the human body [79]. These studies have led to the discovery of a
novel molecular form of albumin and provided a paradigm illustrating how the
function of a protein thiol can be affected by N-homocysteinylation of a protein
lysine residue.
Of the two major physiological forms of human serum albumin (Fig. 5.7 ),
albumin-Cys 34 -S-S-Cys (containing cysteine in a disulfide linkage with Cys 34
of albumin) reacts with Hcy-thiolactone faster than albumin-Cys 34 -SH (mercapto-
albumin, containing Cys 34 with a free thiol). The reactivity of Lys 525 residue,
a predominant site of N-homocysteinylation, is about twofold greater in albumin-
Cys 34 -S-S-Cys than in mercaptoalbumin. The N-homocysteinylated form of