The mechanism of autoprocessing and catalysis in human asparaginase-like protein 1
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Differences in metabolic pathways between cancer and normal tissue can lead to a new generation of protease therapeutics. Heterologous enzymes to deplete specific amino acids that are deficient in cancer cells have applications for treating a number of cancers. One successful example is acute lymphoblastic leukemia (ALL) treatment with bacterial L-asparaginase to systematically deplete L-asparagine (L-Asn). However, the repeated or prolonged therapeutic administration of such enzymes is restricted by their immunogenicity and secondary specificity. An alternative strategy using a human version of asparaginase with higher substrate specificity was not available until the recent identification of human asparaginase-like protein 1 (hASRGL1). In order to provide good guidance for hASRGL1 therapeutic engineering, in this dissertation study, we use a combination of structural biology, kinetics, protein engineering and a novel method of differential scanning fluorometry to study the enzyme mechanism of hASRGL1. Autoprocessing is a critical post-translation modification step for hASRGL1 function. It is intertwined with substrate catalysis which fetters the study of hASRGL1 enzyme mechanism. To effectively tackle this problem and to gain a good control over activation of hASRGL1 for therapeutic application, we circumvented this obstacle by a circularly permuted hASRGL1 that uncoupled the autoprocessing reaction, allowing us to kinetically and structurally characterize this enzyme and the precursor-like, hASRGL1-Thr168Ala variant. In doing so, we revealed that a torsional restraint on the Thr168 side-chain helps drive the intramolecular processing reaction. Cleavage and formation of the active site releases the torsional restriction on Thr168, which is facilitated by a small conserved Gly-rich loop near the active site that allows the conformational changes necessary for activation (Chapter 2). Following that, we further delineated the roles of several critical residues in catalyzing autoprocessing and/or substrate catalysis using the uncoupled system of WT-hASRGL1 and cp-hSARGL1 (Chapter3). Furthermore, we developed a de novo method using differential scanning fluorometry to quantitatively analyze the autoprocessing reaction and, for the first time, identified three distinct molecular complexes during maturation that confirmed the existing of half processed heterotrimer. Finally, we found that the dimer-dimer interface is critical for stabilization of the αβ-βα homodimer and the rest of the autoprocessing reaction through a mutagenesis study.