PDBsum entry 5dnd

Hydrolase

PDB id: 5dnd

Contents

Protein chains

353 a.a.

Ligands

ASN ×4

EDO ×4

Waters ×399

PDB id:

5dnd

Name:

Hydrolase

Title:

Crystal structure of the asn-bound guinea pig l-asparaginase 1 catalytic domain active site mutant t116a

Structure:

L-asparaginase. Chain: a, b, c, d. Engineered: yes. Mutation: yes. Other_details: the first 23 residues are from the tag hexahistidine tag and tev protease cleavage site. They were not removed during purification but are not seen in the structure. ThE C-terminus of the protein was cleaved in the drop and is not seen in the structure despite being present throughout the entirety of the purification.

Source:

Cavia porcellus. Guinea pig. Organism_taxid: 10141. Gene: aspg. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.29Å R-factor: 0.210 R-free: 0.242

Authors:

A.M.Schalk,A.Lavie

Key ref:

A.M.Schalk et al. (2016). Experimental Data in Support of a Direct Displacement Mechanism for Type I/II L-Asparaginases. J Biol Chem, 291, 5088-5100. PubMed id: 26733195 DOI: 10.1074/jbc.M115.699884

Date:

09-Sep-15 Release date: 13-Jan-16

Protein chains

H0W0T5  (H0W0T5_CAVPO) -  asparaginase from Cavia porcellus

Seq: 565 a.a.

Struc: 353 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.5.1.1 - asparaginase.
• Reaction: L-asparagine + H2O = L-aspartate + NH4⁺

L-asparagine + H2O (Bound ligand (Het Group name = ASN) corresponds exactly) = L-aspartate + NH4(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1074/jbc.M115.699884

J Biol Chem 291:5088-5100 (2016)

PubMed id: 26733195

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

A.M.Schalk, A.Antansijevic, M.Caffrey, A.Lavie.

ABSTRACT

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.