Structures of Apo and Product-Bound Human l-Asparaginase: Insights into the Mechanism of Autoproteolysis and Substrate Hydrolysis

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, since 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 Km 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 solved 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 on the precise enzyme-substrate interactions. The new understanding of hASNase3 will serve to guide the design of variants that possess a decreased Km value for asparagine, making the human enzyme a suitable replacement for the bacterial asparaginases in cancer therapy.

Loss of ASRGL1 expression is an independent biomarker for disease-specific survival in endometrioid endometrial carcinoma

“…medisapiens.org), and in uterine cancer the outliers belong to the group with low expression, whereas outliers in colorectal cancer have high expression compared to the majority. Moreover, ASRGL1 is induced by progesterone and 5-alpha-di-hydrotestosterone [34,35], which could partly explain differences between hormone-related EEA and hormoneindependent colorectal cancer and non-endometrioid EC. The human ASRGL1 protein contains 308 amino acids and is activated by autocleavage at amino acid 168 to form an alpha-chain and a betachain (Fig.…”

“…3A), which dimerize into a heterodimer [16]. ASRGL1 is relatively stable in its inactive full-length form [35], and can be detected on the Western blot as a band at 40 kDa. In the overexpression lysate (Fig.…”

Human 60-kDa Lysophospholipase Contains an N-terminal l-Asparaginase Domain That Is Allosterically Regulated by l-Asparagine

“…Subsequently, aliquots were tested for residual L-asparaginase activity by the Nesslerization method (29 main protein has been only poorly characterized in one report (24). Our primary motivation to produce and functionally characterize the N-terminal domain of this protein, which structurally and according to its catalytic in vitro properties significantly resembles E. coli cytoplasmic L-asparaginase (ansA; EcASNase1), originated from our previous work on human enzymes that possess L-asparaginase activity (10,19,21). The discovery, molecular engineering, and in vitro evolution of catalytically efficient human L-asparaginases are thought to lay the basis for the replacement of bacterial L-asparaginases presently used as antileukemia therapeutics despite adverse side effects mainly attributed to their bacterial origins (54).…”

Elucidation of the Specific Function of the Conserved Threonine Triad Responsible for Human l-Asparaginase Autocleavage and Substrate Hydrolysis

“…enzymatically inactive), indicating a very slow autocleavage rate [20, 21]. In contrast, AGA and other studied bacterial ASNase3 enzymes are purified as fully cleaved enzymes (i.e.…”