Biochemical and Biophysical Divergences between Two Escherichia coli l-Asparaginase II Variants: Potential for Using...

Escherichia coli l-asparaginase II (EcA2) is essential for treating Acute Lymphoblastic Leukemia, the most common childhood cancer. This enzyme catalyzes the hydrolysis of l-asparagine or l-glutamine to ammonia and l-aspartate or l-glutamate. The first FDA-approved EcA2 biopharmaceutical, Elspar, was introduced in 1978, followed by other biosimilars. Despite stringent approval criteria, variations in plasmatic activity and therapeutic efficacy persist across different EcA2 preparations, often leading to substandard product notifications. Many studies focus on the EcA2 from the E. coli K12 strain (EcA2-K12), which differs by four amino acids from reference biopharmaceuticals, including Elspar (EcA2-4M). Here, we show that EcA2-4 M has over twice the specific activity on both the hydrolysis of l-asparagine and on human lymphoblast cells compared to EcA2-K12. EcA2-K12 demonstrates 4-fold greater specificity for l-asparagine over l-glutamine, considering their k_cat, but similar K_M toward each amino acid. Interestingly, EcA2-K12 has 3-fold lower affinity for l-aspartate, linked to reduced stabilization of its N-terminal active site loop. Although both variants exhibit indistinguishable thermostability, EcA-K12 shows a higher tendency to oligomerize. We solved the 3D structures of both variants by X-ray crystallography, and normal-mode analysis revealed wider conformational changes in EcAK12's active site. Our data indicate that EcA2-K12 has lower activity due to the higher conformational dynamics of the N-terminal active site loop. Nevertheless, EcA2-K12 is a beneficial alternative or complement to existing therapeutic schemes with EcA2-4M, due to its higher specificity to l-asparagine, which is of fundamental importance since activity on l-glutamine is associated with harmful side effects.