Unraveling the Enigmatic Mechanism of <scp>l</scp>-Asparaginase II with QM/QM Calculations

Gesto, Diana; Cerqueira, Nuno M. F. S. A.; Fernandes, Pedro A.; Ramos, María J.

doi:10.1021/ja310165u

Cited by 57 publications

(93 citation statements)

References 31 publications

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“…Proposed Direct Displacement Mechanism for gpASNase1-Our inability to prove the existence of a covalent intermediate, together with the residual activity of the threonine mutants support the conclusion made by Gesto et al (15) for the use of a direct displacement mechanism by type I/II L-asparaginases. Based on our crystal structures of the T19A, T116A, and K188M gpASNase1 mutants in complex with Asn, we prepared a schematic of the active site that represents the putative Michaelis complex (Fig.…”

Section: Structural Analysis Of Gpasnase1 Active Site Mutants Fails Tosupporting

confidence: 72%

“…Gesto et al (15) suggested that the covalent intermediate observed in the E. coli T89V structure is an artifact because of the presence of the mutation. We reasoned that we could solve this source of uncertainty by having both threonines present and slowing the reaction (to allow for trapping of the covalent intermediate) by some other active site mutation.…”

Section: Structural Analysis Of Gpasnase1 Active Site Mutants Fails Tomentioning

confidence: 99%

“…Using computational methods to study the E. coli type II mechanism, these authors discovered that the energetics are not consistent with the assumed covalent intermediate mechanism but rather with a direct displacement mechanism (15). To shed light on this controversy, we conducted kinetic, mutagenesis, and structural studies on gpASNase1.…”

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confidence: 99%

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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: 72%

Section: Structural Analysis Of Gpasnase1 Active Site Mutants Fails Tomentioning

confidence: 99%

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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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“…Those investigations focused on intersubunit contacts and catalytic loop dynamics to find changes that might increase catalytic rate and enzyme stability. A recent simulation 34 also studied the active site based on the 3ECA structure, which has only 1 water molecule in the active site, resulting in absent networks of hydrogen bonds that stabilize the active site. That paucity of water molecules could be significant.…”

Section: Discussionmentioning

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

162

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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“…2,3 Enzymatic activity depends on the classic 2-step nucleophilic attack mechanism in E coli ASNase, initiated by WAT1355 and the cooperation of the highly conserved amino acids of Thr12-Tyr25-Glu283 and Ser58-Gln59-Thr89-Asp90-Lys162 from subunit A, and Asn248C and Glu283C from subunit C, respectively, which are interconnected by strong hydrogen bonds. 4,5 The Thr12-Lys162-Asp90 group acts to deprotonate a water molecule (WAT1355) which subsequently binds to the substrate. The second triplet, Thr12-Ty25-Glu283, stabilizes the resulting tetrahedral intermediate.…”

mentioning

confidence: 99%

Is glutamine depletion needed in ALL disease?

Avramis

2014

Blood

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Unraveling the Enigmatic Mechanism of l-Asparaginase II with QM/QM Calculations

Cited by 57 publications

References 31 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 glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

Is glutamine depletion needed in ALL disease?

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