Structures of Apo and
Product-Bound Human l‑Asparaginase: Insights into
the Mechanism of Autoproteolysis
and Substrate Hydrolysis
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Abstract
Asparaginases catalyze the hydrolysis of the amino acid
asparagine
to aspartate and ammonia. Bacterial asparaginases are used in cancer
chemotherapy to deplete asparagine from the blood, because several
hematological malignancies depend on extracellular asparagine for
growth. To avoid the immune response against the bacterial enzymes,
it would be beneficial to replace them with human asparaginases. However,
unlike the bacterial asparaginases, the human enzymes have a millimolar <i>K</i><sub>m</sub> value for asparagine, making them inefficient
in depleting the amino acid from blood. To facilitate the development
of human variants suitable for therapeutic use, we determined the
structure of human l-asparaginase (hASNase3). This asparaginase
is an N-terminal nucleophile (Ntn) family member that requires autocleavage
between Gly167 and Thr168 to become catalytically competent. For most
Ntn hydrolases, this autoproteolytic activation occurs efficiently.
In contrast, hASNas3 is relatively stable in its uncleaved state,
and this allowed us to observe the structure of the enzyme prior to
cleavage. To determine the structure of the cleaved state, we exploited
our discovery that the free amino acid glycine promotes complete cleavage
of hASNase3. Both enzyme states were elucidated in the absence and
presence of the product aspartate. Together, these structures provide
insight into the conformational changes required for cleavage and
the precise enzyme–substrate interactions. The new understanding
of hASNase3 will serve to guide the design of variants that possess
a decreased <i>K</i><sub>m</sub> value for asparagine, making
the human enzyme a suitable replacement for the bacterial asparaginases
in cancer therapy