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 K(m) 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 K(m) value for
asparagine, making the human
enzyme a suitable replacement for the bacterial asparaginases in
cancer therapy.