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Aspartoacylase
Aspartoacylase
catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate.
Although the exact role of this enzymatic reaction has not yet been
completely elucidated, the metabolism of NAA appears to be necessary in the
formation of myelin lipids and defects in this enzyme lead to Canavan
disease, a fatal neurological disorder. The low catalytic activity and
inherent instability observed with the Escherichia coli-expressed
form of aspartoacylase (Moore,
et al., 2003) suggested the need for a suitable eukaryotic expression
system that would be capable of producing a fully functional, mature
enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris.
While the expression yields are lower than in E. coli, the purified
enzyme is significantly more stable (Le Coq, et al., 2006).
This enzyme form has the same substrate specificity, but is 150-fold more
active than the E. coli-expressed enzyme. The molecular weight of
the purified enzyme, measured by mass spectrometry, is higher than
predicted, suggesting the presence of some posttranslational modifications.
Deglycosylation of aspartoacylase or mutation at
the glycosylation site causes decreased enzyme stability and diminished
catalytic activity. A carbohydrate component has been removed and
characterized by mass spectrometry. In addition to this carbohydrate
moiety, the enzyme has also been shown to contain one zinc atom per
subunit. Chelation studies to remove the zinc
(table 2) results in a reversible loss of catalytic activity, thus
establishing aspartoacylase as a zinc metalloenzyme. Recent structural
studies have confirmed the presence and the role of zinc in the catalytic
activity.
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Table 2: Effect of Metal Chelation
on Aspartoacylase
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Enzyme form
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Metal content
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S.A. (U/mg)
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S.A. (percent)
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Native
enzyme
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1.30 ± 0.04
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8.9
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100 %
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48 hr treatment
with chelator
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0.31
± 0.02
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2.0
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22 %
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72 hr treatment with chelator
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0.23 ± 0.01
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1.2
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14 %
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Viola Research Group