Do bacterial l-asparaginases utilize a catalytic triad Thr-Tyr-Glu?

Aghaiypour, Khosrow; Wlodawer, Alexander; Łubkowski, J.

doi:10.1016/s0167-4838(01)00270-9

Cited by 47 publications

(52 citation statements)

References 32 publications

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“…This arose because the first catalytic threonine is located in a flexible loop with no obvious means of activation, and it was considered more likely that the second, rigid, threonine would be a better candidate for the first step. 26 However, kinetic and mutagenesis data together with the direct visualization of the covalent enzyme-product intermediate confirm that Thr14 in the flexible loop performs the first nucleophilic attack. 19 We also trapped the covalent enzyme intermediate in the wild-type and AnsA[T162A]-asparagine complexes, and this intermediate does indeed reside on Thr14 in AnsA.…”

Section: Discussionmentioning

confidence: 98%

“…Amidohydrolysis proceeds by two sequential nucleophilic attacks by the paired active site threonine residues. 10,16,18,19,26,37 The first attacking threonine releases ammonia and generates a covalent enzyme-product intermediate. During the second attack, the opposing threonine activates a water molecule for product release.…”

Section: Discussionmentioning

confidence: 99%

“…8,10,16,18,19,26,37 The active site of apo AnsA is shown in Figure 6(a), and the two key threonine residues correspond to Thr14 in the extended loop between β1 and α1, and Thr91 in the loop between β3 and α3. The substrate-binding pocket is occupied by electron density that we interpret as a molecule of glycerol.…”

Section: Insights Into the Ansa Active Sitementioning

confidence: 99%

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Crystal Structure and Allosteric Regulation of the Cytoplasmic Escherichia coli l-Asparaginase I

Yun

Nourse

White

et al. 2007

Journal of Molecular Biology

110

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AnsA is the cytoplasmic asparaginase from Escherichia coli involved in intracellular asparagine utilization. Analytical ultracentifugation and X-ray crystallography reveal that AnsA forms a tetrameric structure as a dimer of two intimate dimers. Kinetic analysis of the enzyme reveals that AnsA is positively cooperative, displaying a sigmoidal substrate dependence curve with an [S](0.5) of 1 mM L-asparagine and a Hill coefficient (n(H)) of 2.6. Binding of L-asparagine to an allosteric site was observed in the crystal structure concomitant with a reorganization of the quarternary structure, relative to the apo enzyme. The carboxyl group of the bound asparagine makes salt bridges and hydrogen bonds to Arg240, while the N(delta2) nitrogen interacts with Thr162. Mutation of Arg240 to Ala increases the [S](0.5) value to 5.9 mM, presumably by reducing the affinity of the site for L-asparagine, although the enzyme retains cooperativity. Mutation of Thr162 to Ala results in an active enzyme with no cooperativity. Transmission of the signal from the allosteric site to the active site appears to involve subtle interactions at the dimer-dimer interface and relocation of Gln118 into the vicinity of the active site to position the probable catalytic water molecule. These data define the structural basis for the cooperative regulation of the intracellular asparaginase that is required for proper functioning within the cell.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

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Crystal Structure and Allosteric Regulation of the Cytoplasmic Escherichia coli l-Asparaginase I

Yun

Nourse

White

et al. 2007

Journal of Molecular Biology

110

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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: 99%

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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“…showed good asparaginase activity at all pH (4,5,6,7,8), maximum being at pH 8. At the same pH Streptococcus species D1 and Bacillus polymyxa also showed maximum enzyme activity.…”

Section: World Journal Of Pharmacy and Pharmaceutical Sciencesmentioning

confidence: 97%

Overview on L-Asparaginase

Silpa¹

2017

WJPPS

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L-asparaginase is a potential anti-tumour enzyme that is known for its ability to reduce the amount of L-asparagine necessary for the tumor cells. It has been used against acute lymphoblastic leukemia and lymphosacoma. This review includes methods for isolation and screening of potential microbes for mass production of enzyme. The clinical use of L-asparaginase has been highlighted. Methods of strain improvement that can increase the efficiency of selected strains have also been described. Further sttempts have been also made to describe the various environmental and nutritional paramaeters affecting the enzyme production. The purification methods and also molecular sequencing of L-asparaginase is also discussed.

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Do bacterial l-asparaginases utilize a catalytic triad Thr-Tyr-Glu?

Cited by 47 publications

References 32 publications

Crystal Structure and Allosteric Regulation of the Cytoplasmic Escherichia coli l-Asparaginase I

Crystal Structure and Allosteric Regulation of the Cytoplasmic Escherichia coli l-Asparaginase I

The catalytic triad of serine peptidases

Overview on L-Asparaginase

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