Site‐specific mutagenesis of <i>Escherichia coli</i> asparaginase II

Wehner, Andreas; Harms, E.; Jennings, Michael P.; Beacham, Ifor R.; Derst, Christian; Bast, Peter; Röhm, Klaus‐Heinrich

doi:10.1111/j.1432-1033.1992.tb17210.x

Cited by 37 publications

(28 citation statements)

References 26 publications

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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

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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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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

Self Cite

112

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“…The assay, which uses L-aspartic acid b-hydroxamate as a substrate, 21 was modified as described in supplemental Methods. Glutaminase enzyme activity was measured using a Glutamate Assay kit (Abcam) according to the manufacturer's instructions (see supplemental Methods for details).…”

Section: Determination Of Asparaginase and Glutaminase Enzyme Activitymentioning

confidence: 99%

The glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

Chan

Lorenzi

Anishkin

et al. 2014

Blood

169

131

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• We used molecular dynamics, saturation mutagenesis, and enzymologic screening to develop a glutaminase-free mutant (Q59L) L-ASP.• We then used Q59L to show that glutaminase activity is not required for L-ASP activity against ASNS-negative cancer cells.L-Asparaginase (L-ASP) is a key component of therapy for acute lymphoblastic leukemia. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. Here, we show that L-ASP's glutaminase activity is not always required for the enzyme's anticancer effect. We first used molecular dynamics simulations of the clinically standard Escherichia coli L-ASP to predict what mutated forms could be engineered to retain activity against asparagine but not glutamine. Dynamic mapping of enzyme substrate contacts identified Q59 as a promising mutagenesis target for that purpose. Saturation mutagenesis followed by enzymatic screening identified Q59L as a variant that retains asparaginase activity but shows undetectable glutaminase activity. Unlike wild-type L-ASP, Q59L is inactive against cancer cells that express measurable asparagine synthetase (ASNS). Q59L is potently active, however, against ASNS-negative cells. Those observations indicate that the glutaminase activity of L-ASP is necessary for anticancer activity against ASNS-positive cell types but not ASNS-negative cell types.Because the clinical toxicity of L-ASP is thought to stem from its glutaminase activity, these findings suggest the hypothesis that glutaminase-negative variants of L-ASP would provide larger therapeutic indices than wild-type L-ASP for ASNS-negative cancers. (Blood. 2014;123(23):3596-3606) Introduction L-Asparaginase (L-ASP) is an enzyme drug used in combination with vincristine and a glucocorticoid (eg, dexamethasone) to treat acute lymphoblastic leukemia (ALL).1,2 We 3-6 and others 7 have reported a rationale for testing L-ASP against low-asparagine synthetase (ASNS) solid tumors as well. L-ASP's primary known enzymatic activity is deamidation of asparagine to aspartic acid and ammonia, but it also deamidates glutamine to glutamic acid and ammonia, although with lower affinity and lower maximal rate. L-ASP therapy is often limited by toxic side effects that are generally attributed to the glutaminase activity. 8,9 Those side effects often preclude completion of the full treatment regimen, resulting in poor outcome. 10 The question that arises, however, is whether the therapeutic index of L-ASP could be increased by decreasing its glutaminase activity 8,9 or whether that would also decrease the anticancer effect commensurately.One side of the debate hypothesizes that L-ASP's therapeutic index can be improved by increasing glutaminase activity. In support of that hypothesis, data collected over the last decade have suggested that glutaminase activity generally increases the efficacy of L-ASP and is sometimes required to achieve an anticancer effect. Those studies have reported asparaginase activity to be expendable. [11][12][13][...

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“…Each active site consists of side chains belonging to two domains of adjacent subunits. From the structures of EcA and ErA and from the available enzymatic data (Bagert & R6hm, 1989;Wehner et al, 1992;Derst, Hensling & R6hm, 1992), the following residues (with EcA numbering in parentheses) appear to be crucial for activity: Thrl 2(12), Tyr26(25), Ser59(58), Thr92(89), Asp93(90), Ser117(114), Lys165(162) in one subunit, and Ser252(248) and Glu288(283) from the adjacent subunit. These residues are similarly arranged in the structures of EcA, ErA and AGA.…”

Section: Active Sites Of Amidohydrolasesmentioning

confidence: 99%

Refined crystal structure of Acinetobacter glutaminasificans glutaminase–asparaginase

Łubkowski¹,

Wlodawer²,

Housset³

et al. 1994

Acta Cryst D

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The crystal structure of glutaminase-asparaginase from Acinetobacter glutaminas~'cans has been reinterpreted and refined to an R factor of 0.171 at 2.9 A resolution, using the same X-ray diffraction data that were used to build a preliminary model of this enzyme [Ammon, Weber, Wlodawer, Harrison, Gilliland, Murphy, Sj61in & Roberts (1988). J. Biol. Chem. 263,[150][151][152][153][154][155][156]]. The current model, which does not include solvent, is based in part on the related structure of Escherichia coli asparaginase and is significantly different from the structure of the enzyme from A. glutaminasificans described previously. The reason for the discrepancies has been traced to insufficient phasing power of the original heavyatom derivative data, which could not be compensated for fully by electron-density modification techniques. The corrected structure of A. glutaminasificans glutaminase-asparaginase is presented and compared with the preliminary model and with the structure of E. coli asparaginase.

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Site‐specific mutagenesis of Escherichia coli asparaginase II

Cited by 37 publications

References 26 publications

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

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

The glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

Refined crystal structure of Acinetobacter glutaminasificans glutaminase–asparaginase

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