ArticleJournal of molecular biology2014
Elucidation of the specific function of the conserved threonine triad responsible for human L-asparaginase autocleavage and substrate hydrolysis.
Article in Journal of molecular biology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed, 40 citations in OpenAlex.
- Overcoming challenges in the production of a human-like L-asparaginase: advanced strategies for expression and purification.Biotechnology reports (Amsterdam, Netherlands) · 2026Article
- Stereochemical aspects of the nucleophilic attack in different classes of L-asparaginases.IUCrJ · 2026Review
- Towards a dependable data set of structures for L-asparaginase research.Acta crystallographica. Section D, Structural biology · 2024Article
- Analysis ofHeliyon · 2024Article
- Biochemical characterization of L-asparaginase isoforms fromFrontiers in chemistry · 2024Article
- Elucidation of the Active Form and Reaction Mechanism in Human Asparaginase Type III Using Multiscale Simulations.Journal of chemical information and modeling · 2023Article
- The effects of nature-inspired amino acid substitutions on structural and biochemical properties of the E. coli L-asparaginase EcAIII.Protein science : a publication of the Protein Society · 2023Article
- In Silico Design of a Chimeric Humanized L-asparaginase.International journal of molecular sciences · 2023Article
- Insights into the Distribution and Functional Properties of l-Asparaginase in the Archaeal Domain and Characterization ofACS omega · 2022Article
- Structural and biophysical studies of new L-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase.Acta crystallographica. Section D, Structural biology · 2022Article
- Anticancer Activity of Extremely Effective Recombinant L-Asparaginase fromJournal of microbiology and biotechnology · 2022Article
- Structural Aspects ofInternational journal of molecular sciences · 2022Article
- Review
- Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions.Pharmaceutics · 2022Review
- Structural and biophysical aspects of l-asparaginases: a growing family with amazing diversity.IUCrJ · 2021Review
- Nucleic Acid Delivery with Red-Blood-Cell-Based Carriers.International journal of molecular sciences · 2021Review
- Targeting the Proline-Glutamine-Asparagine-Arginine Metabolic Axis in Amino Acid Starvation Cancer Therapy.Pharmaceuticals (Basel, Switzerland) · 2021Review
- Bacillus sonorensis L. Asparaginase: Cloning, Expression in E. coli and Characterization.The protein journal · 2020Article
- Improving the Treatment of Acute Lymphoblastic Leukemia.Biochemistry · 2020Article
- Enzymes in Metabolic Anticancer Therapy.Advances in experimental medicine and biology · 2019Review
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
Our long-term goal is the design of a human l-asparaginase (hASNase3) variant, suitable for use in cancer therapy without the immunogenicity problems associated with the currently used bacterial enzymes. Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. The key property allowing for the depletion of blood asparagine by bacterial asparaginases is their low micromolar KM value. In contrast, human enzymes have a millimolar KM for asparagine. Toward the goal of engineering an hASNase3 variant with micromolar KM, we conducted a structure/function analysis of the conserved catalytic threonine triad of this human enzyme. As a member of the N-terminal nucleophile family, to become enzymatically active, hASNase3 must undergo autocleavage between residues Gly167 and Thr168. To determine the individual contribution of each of the three conserved active-site threonines (threonine triad Thr168, Thr186, Thr219) for the enzyme-activating autocleavage and asparaginase reactions, we prepared the T168S, T186V and T219A/V mutants. These mutants were tested for their ability to cleave and to catalyze asparagine hydrolysis, in addition to being examined structurally. We also elucidated the first N-terminal nucleophile plant-type asparaginase structure in the covalent intermediate state. Our studies indicate that, while not all triad threonines are required for the cleavage reaction, all are essential for the asparaginase activity. The increased understanding of hASNase3 function resulting from these studies reveals the key regions that govern cleavage and the asparaginase reaction, which may inform the design of variants that attain a low KM for asparagine.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.