Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.
Biochemistry. 2012 Aug 28;51(34):6816-26. doi: 10.1021/bi300870g. Epub 2012 Aug 14.
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.
天冬酰胺酶催化氨基酸天冬酰胺水解为天冬氨酸和氨。细菌天冬酰胺酶用于癌症化疗以从血液中耗尽天冬酰胺，因为几种血液系统恶性肿瘤依赖细胞外天冬酰胺生长。为了避免针对细菌酶的免疫反应，用人类天冬酰胺酶替代它们将是有益的。然而，与细菌天冬酰胺酶不同，人类酶对天冬酰胺的毫摩尔 K(m) 值，使得它们从血液中耗尽氨基酸的效率较低。为了促进适合治疗用途的人类变体的开发，我们确定了人 l-天冬酰胺酶 (hASNase3) 的结构。这种天冬酰胺酶是 N-末端亲核体 (Ntn) 家族成员，需要甘氨酸 167 和苏氨酸 168 之间的自切割才能成为催化有效。对于大多数 Ntn 水解酶，这种自动蛋白水解激活作用效率很高。相比之下，hASNas3 在未切割状态下相对稳定，这使我们能够在切割之前观察到酶的结构。为了确定切割状态的结构，我们利用我们的发现，即游离氨基酸甘氨酸促进 hASNase3 的完全切割。在没有和存在产物天冬氨酸的情况下阐明了这两种酶状态。这些结构共同提供了对切割所需的构象变化以及精确的酶-底物相互作用的深入了解。对 hASNase3 的新认识将有助于指导设计具有降低的天冬酰胺 K(m) 值的变体，从而使人类酶成为癌症治疗中细菌天冬酰胺酶的合适替代品。
