A catalytic role for threonine‐12 of <i>E. coli</i> asparaginase II as established by site‐directed mutagenesis

Harms, E.; Wehner, Andreas; Aung, Hnin-Pwint; Röhm, Klaus‐Heinrich

doi:10.1016/0014-5793(91)80723-g

Cited by 36 publications

(32 citation statements)

References 16 publications

(2 reference statements)

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“…In addition, our results that reveal significant residual activity for the T19A mutant are consistent with experiments performed on the E. coli type II L-asparaginase, where mutation of the presumed covalent intermediate-forming threonine reduced the rate by only 15,000-fold (10). Together, these mutagenesis experiments, by revealing L-asparaginases that have higher than expected residual activity upon mutation of their active site threonine to alanine, question the long held assumption of a ping-pong mechanism.…”

Section: Structural Analysis Of Gpasnase1 Active Site Mutants Fails Tosupporting

confidence: 78%

“…Notably, the authors concede that "The present oxygen exchange experiments provide results that are consistent with, but do not require, the involvement of a covalent intermediate " (9). Biochemical studies (10,11) and crystal structures of L-asparaginase (12, 13) identified Thr 12 and Thr 89 as putative active site residues that would form such a covalent intermediate, consis- tent with the conservation of these threonine residues in type I/II L-asparaginases. Mutagenesis studies of the E. coli type II enzyme revealed that the T12A mutant is still active, albeit with 0.01% (10) to 0.04% (11) activity relative to the wild type enzyme.…”

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confidence: 48%

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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 Tosupporting

confidence: 78%

mentioning

confidence: 48%

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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“…The oligonucleotide-directed mutagenesis of the ansB gene in M13mpl9 [17,18] and the overexpression of EtA [19] have been described in detail elsewhere. Mutants T89V and T89S were constructed via the degenerate oligonucleotide 5'-ACC CAC GGT (G,A)(T,G)C GAC ACG ATG-3'.…”

Section: Methodsmentioning

confidence: 99%

A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

Palm¹,

Łubkowski²,

Derst³

et al. 1996

FEBS Letters

112

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Escherichia coli asparaginase |I catalyzes the hydrol-) sis of L-asparagine to L-aspartate via a threonine-bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threonines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 A resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.:£ey words'." Asparaginase II; Acyl-enzyme intermediate; !'hreonine amidohydrolase; Enzymatic mechanism !. IntroductionAsparaginases catalyze the hydrolysis of L-asparagine to L~spartate and ammonia. The enzymes isolated from Escheri-'hia coli (EcA) and Erwinia chrysanthemi (ERA) have been ltilized as anti-leukemia drugs for many years [1]. Treatment vith asparaginases is often accompanied by severe side effects, vhich are partially attributed to the glutaminase activity of hese enzymes [2]. In order to understand the enzyme specifi-:ity and to ultimately eliminate the glutaminase activity, the nechanism of action must be elucidated. Several members of t larger family of homologous L-asparaginases have thus been horoughly investigated over many years.Results of kinetic measurements [3,4] indicated that the en-,ymatic reaction proceeds via a covalent intermediate, probtbly a ~-aspartyl enzyme (Fig. 1). This mechanism was con-,irmed by NMR studies with aspartate through the oxygen ~xchange reaction with bulk solvent at the side chain carboxdate [5]. These experiments showed that at pH < 5 L-aspartate ~ith a protonated side chain binds to the enzyme as tightly as ~-asparagine and it can also form an acyl-enzyme intermediate, subsequently hydrolyzed to L-aspartate. Thus, at low pH 3oth L-aspartate and L-asparagine can function as substrates. l'he nature and identity of the primary nucleophile of the mzyme participating in the formation of the acyl-enzyme in-*Corresponding author. Fax: (1) . Each has the same tetrameric quaternary structure as it has in solution, a homotetramer of approx. 4x330 residues. The tetramer is more accurately described as a dimer of dimers. Each of two identical active sites in each dimer is formed by both monomers. In the structure the active sites have been observed with and without aspartate as ligand. Part of the active site is covered by a flexible loop that contains the two important residues Thr-12 and Tyr-25. The hydroxyl groups of Thr-12 and Thr-89 are closest to the side chain carboxylate of an aspartate bound in the active site. These residues are the most likely candidates for the primary nucleophile. Bacterial L-asparaginases were the first threonine amidohydrolases described in the literature. Only recently, two other threonine amidohydrolases, 20S proteasome [14] and aspartylglucosaminidase [15], have been reported. Both enzymes belong t...

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“…The type II enzyme with the high affinity markedly inhibited lymphomas in mice, whereas the type I enzyme with the low affinity was ineffective (Schwarts et al, 1966). Their properties have been studied in Escherichia coli (Bagert and Röhm, 1989;Harms et al, 1991aHarms et al, , 1991b, Erwinia carotovora (Kotzia and Labrou, 2005), Erwinia chrythanthemi (Aghaiypour et al, 2001), Pseudomonas aeruginosa (Geckil et al, 2006), Saccharomyces cerevisiae (Kim et al, 1988), and Pyrococcus horikoshii (Yao et al, 2005). Especially, the type II enzymes of Escherichia coli and Erwinia chrysanthemi have been intensively investigated with respect to their antitumor activity.…”

Section: Introductionmentioning

confidence: 99%

Overexpression of type I L-asparaginase ofBacillus subtilis inEscherichia coli, rapid purification and characterisation of recombinant type I L-asparaginase

et al. 2008

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The present study focused on cloning and expression of the ansA gene, encoding putative asparaginase I of Bacillus subtilis in Escherichia coli, and investigation of the basic properties of the recombinant enzyme for application in food processing industry. The ansA gene of B. subtilis was cloned by polymerase chain reaction and expressed in E. coli Rosseta Gami B. It consisted of an open reading frame of 987 nucleotides encoding a protein of 329 amino acids with a calculated molecular mass of 36441 Da. The recombinant enzyme showed increased expression (21.7 U mg -1 ) and was purified to homogeneity by a two-step procedure that included L-asparagine affinity column chromatography. Based on the kinetic properties and primary structure of the native enzyme, the product of the ansA gene was classified as L-asparaginase I. The enzyme showed a high specific activity compared with that of L-asparaginase II from E. coli. Results of this study will allow us to apply the enzyme to food industry.

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A catalytic role for threonine‐12 of E. coli asparaginase II as established by site‐directed mutagenesis

Cited by 36 publications

References 16 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

A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

Overexpression of type I L-asparaginase ofBacillus subtilis inEscherichia coli, rapid purification and characterisation of recombinant type I L-asparaginase

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