Atomic resolution structure of<i>Erwinia chrysanthemi</i><scp>L</scp>-asparaginase

Łubkowski, J.; Dauter, Mirosława; Aghaiypour, Khosrow; Wlodawer, Alexander; Dauter, Zbigniew

doi:10.1107/s0907444902019443

Cited by 56 publications

(57 citation statements)

References 29 publications

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“…The Asn side chain amino group forms a hydrogen bond with the Ala 142 carbonyl group, whereas the oxygen atom present in the product Asp is repulsed by the Ala 142 carbonyl group, resulting in the increased separation observed in the Asp complex structure. The repulsive interaction between the Asp and carbonyl of Ala 142 (a conserved alanine) has been attributed as a mechanism to remove the product from the active site (22). Additionally, this analysis provides a structural explanation for the need for low pH to obtain tight Asp binding (12,14,23); at low pH, the carboxylate side chain of Asp would be partially protonated, negating the repulsive effects of the Ala 142 carbonyl.…”

Section: Structural Analysis Of Gpasnase1 Active Site Mutants Fails Tomentioning

confidence: 92%

Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

Schalk

Antansijevic

Caffrey

et al. 2016

Journal of Biological Chemistry

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Bacterial L-asparaginases play an important role in the treatment of certain types of blood cancers. We are exploring the guinea pig L-asparaginase (gpASNase1) as a potential replacement of the immunogenic bacterial enzymes. The exact mechanism used by L-asparaginases to catalyze the hydrolysis of asparagine into aspartic acid and ammonia has been recently put into question. Earlier experimental data suggested that the reaction proceeds via a covalent intermediate using a ping-pong mechanism, whereas recent computational work advocates the direct displacement of the amine by an activated water. To shed light on this controversy, we generated gpASNase1 mutants of conserved active site residues (T19A, T116A, T19A/T116A, K188M, and Y308F) suspected to play a role in hydrolysis. Using x-ray crystallography, we determined the crystal structures of the T19A, T116A, and K188M mutants soaked in asparagine. We also characterized their steady-state kinetic properties and analyzed the conversion of asparagine to aspartate using NMR. Our structures reveal bound asparagine in the active site that has unambiguously not formed a covalent intermediate. Kinetic and NMR assays detect significant residual activity for all of the mutants. Furthermore, no burst of ammonia production was observed that would indicate covalent intermediate formation and the presence of a ping-pong mechanism. Hence, despite using a variety of techniques, we were unable to obtain experimental evidence that would support the formation of a covalent intermediate. Consequently, our observations support a direct displacement rather than a ping-pong mechanism for L-asparaginases.Currently, there still exists a gap in knowledge regarding the enzymatic mechanism by which L-asparaginases hydrolyze the amino acid L-asparagine to L-aspartic acid and ammonia (Fig. 1A). L-Asparaginases have garnered special attention because of their clinical use in the treatment of certain blood cancers (1). In addition, L-asparaginases are also used commercially by the food industry for reducing the production of acrylamide (2), a compound being investigated for its toxicity and carcinogenicity in humans. Hence, understanding the enzymatic mechanism of these important enzymes can be used to inform the design of variants with improved properties. Our specific interest is with the guinea pig L-asparaginase type I enzyme, which holds potential as a replacement for the currently clinically used bacterial enzymes (see more below).L-Asparaginases can be divided, based on sequence and structural homology, into two unrelated families: type I and II L-asparaginases belong to one family, and type III enzymes belong to another (3). The type I and II enzymes are very similar in terms of overall structure and conservation of active site residues (3). In Escherichia coli, the type I enzyme is cytoplasmic, and type II is periplasmic (3). In addition to having different cellular localization, bacterial type II enzymes have a much lower K m value for Asn (low micromolar versus millimolar). This ϳ...

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Section: Structural Analysis Of Gpasnase1 Active Site Mutants Fails Tomentioning

confidence: 92%

Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

Schalk

Antansijevic

Caffrey

et al. 2016

Journal of Biological Chemistry

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“…Asparaginases, which catalyze the hydrolysis of Lasparagine to L-aspartate and ammonia, contain a threonine nucleophile instead of serine. While the crystal structure was determined for the E. coli [117,118] and the Erwinia chrysanthemi [119,120] L-asparaginases, the enzymatic mechanism is unclear [121]. The enzyme displays an unusual active site that exhibits two essential threonine residues, and either appears to be a good candidate for functioning as nucleophile.…”

Section: Further Variations In the Members Of The Catalytic Triadmentioning

confidence: 98%

The catalytic triad of serine peptidases

Polgár

2005

Cell. Mol. Life Sci.

391

311

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The catalytic action of serine peptidases depends on the interplay of a nucleophile, a general base and an acid. In the classic trypsin and subtilisin families this catalytic triad is composed of serine, histidine and aspartic acid residues and exhibits similar spatial arrangements, but the order of the residues in the amino acid sequence is different. By now several new families have been discovered, in which the nucleophile-base-acid pattern is generally conserved, but the individual components can vary. The variations illustrate how different groups and different protein structures achieve the same reaction.

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“…Although these glycosylations are not observed in the active or substratebinding sites, enzyme activity may be modified by an allosteric effect. This probable allosteric effect would be possible-and may alter the proposed conformation as given previously and changing pharmacological properties and contribute to side effects [14,15].…”

Section: Narkhyun Bae Arnold Pollak Gert Lubecmentioning

confidence: 94%

Proteins from Erwinia asparaginase Erwinase^® and E. coli asparaginase 2 MEDAC^® for treatment of human leukemia, show a multitude of modifications for which the consequences are completely unclear

2011

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L-Asparaginase from Erwinia chrysanthemi (ASPG_ERWCH; UniProtKB accession number P06608 (Erwinase(®))) and L-asparaginase 2 from Escherichia coli (ASPG2_ECOLI; UniProtKB accession number P00805 (Medac(®))), both L-asparagine amidohydrolases, are widely used for the treatment of acute lymphoblastic leukemia. A series of serious side effects have been reported and this warrants studies into the protein chemistry of the medical products sold. Mass spectrometry (MS) data on ASPG_ERWCH and ASPG2_ECOLI have not been published so far and herein a gel-based proteomics study was performed to provide information about sequence and modifications of the commercially available medical products. ASPG_ERWCH and ASPG2_ECOLI were applied onto two-dimensional gel electrophoresis, spots were in-gel digested with several proteases and resulting peptides and protein modifications were analysed by nano-ESI-LC-MS/MS. Four spots were observed for ASPG_ERWCH, six spots were observed for ASPG2_ECOLI and the identified proteins showed high sequence coverage without sequence conflicts. Several protein modifications including technical and posttranslational modifications were demonstrated. Protein modifications are known to change physicochemical, immunochemical, biological and pharmacological properties and results from this work may challenge re-designing of the product including possible removal of the modifications by the manufacturer because it is not known whether they are contributing to the serious adverse effects of the protein drug.

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Atomic resolution structure ofErwinia chrysanthemiL-asparaginase

Cited by 56 publications

References 29 publications

Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

The catalytic triad of serine peptidases

Proteins from Erwinia asparaginase Erwinase^® and E. coli asparaginase 2 MEDAC^® for treatment of human leukemia, show a multitude of modifications for which the consequences are completely unclear

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Atomic resolution structure ofErwinia chrysanthemiL-asparaginase

Cited by 56 publications

References 29 publications

Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

The catalytic triad of serine peptidases

Proteins from Erwinia asparaginase Erwinase® and E. coli asparaginase 2 MEDAC® for treatment of human leukemia, show a multitude of modifications for which the consequences are completely unclear

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Product

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Proteins from Erwinia asparaginase Erwinase^® and E. coli asparaginase 2 MEDAC^® for treatment of human leukemia, show a multitude of modifications for which the consequences are completely unclear