Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 45

2 diabetes, plasma Hcy-thiolactone concentrations are negatively correlated with
HDL levels (r ¼ 0.037) [362], consistent with the ability of

HDL-associated PON1 protein to hydrolyze Hcy-thiolactone [81]. Taken together,
these results provide evidence that PON1 has the ability to protect proteins against
N-homocysteinylation both in vitro and in vivo in humans.
PON1 is the only enzyme known to metabolize Hcy-thiolactone in human and
other mammalian sera, with the highest levels observed in rabbits, which have six
times higher Hcy-thiolactonase/PON1 activity than that present in an average
human serum [153]. However, serum Hcy-thiolactonase activity is absent in
birds, including chicken [81, 153], which makes chicken embryos particularly
sensitive to Hcy-thiolactone toxicity [154, 155].
Substrate specificity studies of purified human serum Hcy-thiolactonase/PON1
show that L -Hcy-thiolactone is a preferred natural substrate [81]. D -Hcy-thiolactone
and

0.223, P ¼

-thiobutyrolactone are hydrolyzed at a rate fourfold slower and 5.5-fold faster,
respectively, than L -Hcy-thiolactone by the enzyme [85]. The artificial substrates
phenyl acetate and paraoxon (hydrolyzed 2,800 and 3.3 times faster than L -Hcy-
thiolactone) are noncompetitive inhibitors of the Hcy-thiolactonase activity
suggesting that Hcy-thiolactone, phenyl acetate, and paraoxon are hydrolyzed at
different sites of the enzyme [81]. This suggestion is supported by structure/
function studies showing that specific active sites mutations have different effects
on arylesterase, paraoxonase, and lactonase activities of the PON1 protein [263,
264]. Other inhibitors of the Hcy-thiolactonase activity such as isoleucine and
penicillamine are also noncompetitive, suggesting the presence of a distinct
amino acid-binding effector site on PON1 [81]. Hcy-thiolactonase and paraoxonase
activities are strongly correlated in various populations [152, 250, 261], indicating
that the artificial paraoxonase activity is a good surrogate for the natural Hcy-
thiolactonase activity of the PON1 protein.
Hcy-thiolactone metabolism is impaired in Pon1 / mice. For example, serum
Hcy-thiolactonase activity is absent in Pon1 / mice [81, 140, 153]. Pon1 / mice
have significantly elevated brain Hcy-thiolactone and excrete more Hcy-thiolactone
in urine compared with Pon1 +/+ littermates [140]. Furthermore, significantly less
plasma Hcy is generated from intraperitoneally injected Hcy-thiolactone in Pon1 /
mice than in Pon1 +/+ animals.
Pon1 / mice are more susceptible than wild-type littermates to neurotoxicity
induced by intraperitoneal injections of Hcy-thiolactone (Table 3.10 ) [140]. Fol-
lowing intraperitoneal injections of Pon1 / mice and Pon1 +/+ littermates with
convulsant doses of L -Hcy-thiolactone (3,700 nmol g 1 body weight), all mice
become somnolescent within 5-10 min. Convulsions, characterized by spontaneous
tonic-clonic, grand mal seizures (kangaroo position, extension of fore and hind
limbs and tail, status epilepticus), and running fits, occur within 50 min [140]. The
incidence of seizures is significantly increased in Pon1 / mice compared with
Pon1 +/+ animals (52.8 vs. 29.5 %, P <

γ

0.042) (Table 3.10 ). Seizure latency (i.e.,
time to first seizure) is significantly decreased for Pon1 / mice compared with
Pon1 +/+ animals (33.8 min vs. 41.2 min, P ¼ 0.019). While only one mouse out

of the 44 Pon1 +/+ mice (2.3 %) dies (at 61 min) after L -Hcy-thiolactone injection,