Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 26

The characteristic UV absorption at 240 nm disappears during the reaction of
Hcy-thiolactone with amines, which facilitates studies of the N-homocysteinylation
reaction. The rate of Hcy-thiolactone reaction with lysine at pH 7.4, measured
spectroscopically at 240 nm, is first order with respect to both Hcy-thiolactone and
lysine. The rate increases with increasing pH, consistent with the lysine base form
(with uncharged amino groups) being the reacting species. The second-order rate
constant's dependence on pH (Fig. 3.3b ) is consistent with ionization of Hcy-
thiolactone with pK a ¼

6.67 and suggests that the positively charged acid form of
Hcy-thiolactone is more reactive with lysine (k obs /[Lys base] ¼ 11,870 M 1 h 1 )
than the neutral base form (k obs /[Lys base] ¼ 1,500 M 1 h 1 ) (Table 3.2 ). The
reaction of Hcy-thiolactone with purified human serum albumin exhibits similar pH
dependence (Fig. 3.3c ) and indicates that the acid form of Hcy-thiolactone is
191-fold more reactive with the protein than the base form (764.0 vs. 4.0 M 1 h 1 ,
Table 3.2 ).

3.2.5 Reactivity Toward Carbonyl Compounds

The -amino group of Hcy-thiolactone is highly reactive toward electrophiles, such

as formaldehyde, acetaldehyde, pyridoxal phosphate, o-phthalaldehyde, or strepto-
mycin (Tables 3.2 and 3.4 ) [68, 84]. Corresponding 1,3-tetrahydrothiazine-4-car-
boxylic acids are formed as products of these reactions (Reaction 3.5 ) [84]. The rate
of condensation of Hcy-thiolactone with a large molar excess of formaldehyde or
acetaldehyde, measured spectrophotometrically at 240 nm, is first order with
respect to Hcy-thiolactone. The dependence of the pseudo-first-order rate constant
on aldehyde concentration is more complex: at low concentration of aldehyde, the
rate constant increases linearly with the aldehyde concentration, but at high
concentrations, the rates level off and are less dependent on aldehyde concentration.
Such kinetic behavior indicates formation of an intermediate (Reaction 3.5 ). A
similar

α

-amino group of Hcy-thiolactone and the
tyrosylquinone cofactor of lysine oxidase in vitro causes irreversible inactivation
of the enzyme [216].
Changes in pH affect the ionization status of the

reaction between the α

-amino group of Hcy-
thiolactone and the rate of its reaction with aldehydes (Fig. 3.3d ). For example,
the positively charged acid form of Hcy-thiolactone reacts with formaldehyde and
acetaldehyde 79 and 73 times faster, respectively (Table 3.2 ), than the neutral base
form (structures of the acid and base forms are shown in Reaction 3.3 ) [84]. At
physiological pH

α

7.4, second-order rate constants for the reaction with
aldehydes are 1,200-8,648 M 1 h 1 (Table 3.4 ) more than 200 times faster than
for the reaction of Hcy-thiolactone with lysine (Table 3.3 ).
The facile formation of tetrahydrothiazines from Hcy-thiolactone and aldehydes
(Reaction 3.5 ) raises an interesting possibility that the endogenous Hcy-thiolactone
formed in the human body may be inactivated and disposed of in the form of

¼