Sensitive and Rapid Assay for L-Asparaginase

Frohwein, Yaacov Zvi; Friedman, Mark L.; Reizer, Jonathan; Grossowicz, N.

doi:10.1038/newbio230158a0

Cited by 21 publications

(10 citation statements)

References 3 publications

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“…6 Hydrolysis of AHA yields not only L-Asp but also hydroxylamine, which is easily detected. 17 We note that the leaving groups of both L-Asn and AHA will require protonation during hydrolysis, but their respective p K a values differ greatly (i.e., NH 4 + ↔ NH 3 , p K a = 9.25; NH 3 + –OH ↔ NH 2 –OH, p K a = 5.96 18 ). Thus, at physiological pH values, hydroxylamine is a stable leaving group and unlike ammonia does not require an extra protonation step.…”

Section: Resultsmentioning

confidence: 83%

Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages

Irani

Crutchfield

et al. 2016

Biochemistry

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The human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal nucleophile (Ntn) family that hydrolyzes L-asparagine and isoaspartyl-dipeptides. The nascent protein folds into an αβ–βα sandwich fold homodimer that cleaves its own peptide backbone at the G167–T168 bond, resulting in the active form of the enzyme. However, biophysical studies of hASRGL1 are difficult because of the curious fact that intramolecular cleavage of the G167–T168 peptide bond reaches only ≤50% completion. We capitalized upon our previous observation that intramolecular processing increases thermostability and developed a differential scanning fluorimetry assay that allowed direct detection of distinct processing intermediates for the first time. A kinetic analysis of these intermediates revealed that cleavage of one subunit of the hASRGL1 subunit drastically reduces the processing rate of the adjacent monomer, and a mutagenesis study showed that stabilization of the dimer interface plays a critical role in this process. We also report a comprehensive analysis of conserved active site residues and delineate their relative roles in autoprocessing and substrate hydrolysis. In addition to glycine, which was previously reported to selectively accelerate hASRGL1 cleavage, we identified several novel small molecule activators that also promote intramolecular processing. The structure–activity analysis supports the hypothesis that multiple negatively charged small molecules interact within the active site of hASRGL1 to act as a base in promoting cleavage. Overall, our investigation provides a mechanistic understanding of the maturation process of this Ntn hydrolase family member.

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Section: Resultsmentioning

confidence: 83%

Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages

Irani

Crutchfield

et al. 2016

Biochemistry

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“…However, this method has certain limitations such low sensitivity and the fact that it do not measure intracellular ASNase activity (Vaishali and Bhupendra, 2017). After the initial screening, other methods are required for the quantitative evaluation of enzymatic activity such as the aforementioned Nessler's reaction (Peterson and Ciegler, 1969); the L-aspartyl-β-hydroxamic acid (AHA) method, wich evaluates aspartyl β-hydroxamate formation after L-asparagine hydrolysis in the presence of hydroxylamine (Frohweinm et al, 1971); circular dichroism spectroscopy (Kudryashova and Sukhoverkov, 2016); amplex Red method (Karamitros et al, 2014), among others. The screening methods for finding enzymes with reduced glutaminase activity are similar to the plate methods used for ASNase activity detection, i.e., they are based on pH switch, but use GLN instead of ASN as a substrate.…”

Section: L-asparaginase Manufacturing: Production Process and Purificmentioning

confidence: 99%

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

Brumano

Silva

Costa-Silva

et al. 2019

Front. Bioeng. Biotechnol.

144

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L-Asparaginase (ASNase) is a vital component of the first line treatment of acute lymphoblastic leukemia (ALL), an aggressive type of blood cancer expected to afflict over 53,000 people worldwide by 2020. More recently, ASNase has also been shown to have potential for preventing metastasis from solid tumors. The ASNase treatment is, however, characterized by a plethora of potential side effects, ranging from immune reactions to severe toxicity. Consequently, in accordance with Quality-by-Design (QbD) principles, ingenious new products tailored to minimize adverse reactions while increasing patient survival have been devised. In the following pages, the reader is invited for a brief discussion on the most recent developments in this field. Firstly, the review presents an outline of the recent improvements on the manufacturing and formulation processes, which can severely influence important aspects of the product quality profile, such as contamination, aggregation and enzymatic activity. Following, the most recent advances in protein engineering applied to the development of biobetter ASNases (i.e., with reduced glutaminase activity, proteolysis resistant and less immunogenic) using techniques such as site-directed mutagenesis, molecular dynamics, PEGylation, PASylation and bioconjugation are discussed. Afterwards, the attention is shifted toward nanomedicine including technologies such as encapsulation and immobilization, which aim at improving ASNase pharmacokinetics. Besides discussing the results of the most innovative and representative academic research, the review provides an overview of the products already available on the market or in the latest stages of development. With this, the review is intended to provide a solid background for the current product development and underpin the discussions on the target quality profile of future ASNase-based pharmaceuticals.

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“…Enzymatic decomposition of aspartic acid-q-hydroxamate and glutamic acid-y-monohydraxamate was determined by the method of Frohwein (6).…”

Section: Methodsmentioning

confidence: 99%

Purification and properties of L-glutaminase-L-asparaginase from Pseudomonas acidovorans

Davidson¹,

Brear²,

Wingard³

et al. 1977

J Bacteriol

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An enzyme that catalyzes the hydrolysis of both glutamine and asparagine has been purified to homogeneity from extracts of Pseudomonas acidovorans. The enzyme having a ratio of glutaminase to asparaginase of 1.45:1.0 can be purified by a relatively simple procedure and is stable upon storage. The glutaminase-asparaginase has a relatively high affinity for-asparagine (K,,, = 1.5 x 10-5 M) and L-glutamine (K,, = 2.2 x 10-5 M) and has a molecular weight of approximately 156,000, the subunit molecular weight being approximately 39,000. Injections of the enzyme produced only slight increases in the survival time of C3H/HE mice carrying the asparagine-requiring 6C3HED Gardner lymphoma and of white Swiss mice carrying the glutamine-requiring Ehrlich lymphoma.'

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Sensitive and Rapid Assay for L-Asparaginase

Cited by 21 publications

References 3 publications

Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages

Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

Purification and properties of L-glutaminase-L-asparaginase from Pseudomonas acidovorans

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