PDBsum entry 6v2c

Hydrolase

PDB id: 6v2c

Contents

Protein chains

327 a.a.

Waters ×965

PDB id:

6v2c

Name:

Hydrolase

Title:

Complex of mutant (k162m) of e. Coli l-asparaginase ii with l-asp. Covalent acyl-enzyme intermediate and tetrahedral intermediate

Structure:

L-asparaginase 2. Chain: a, c, b, d. Synonym: l-asparaginase ii,l-asnase ii,l-asparagine amidohydrolase ii. Engineered: yes. Mutation: yes

Source:

Escherichia coli (strain k12). Organism_taxid: 83333. Strain: k12. Gene: ansb, b2957, jw2924. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Expression_system_variant: jc2. Expression_system_cell: mesophilic bacteria.

Resolution:

2.00Å R-factor: 0.173 R-free: 0.235

Authors:

J.Lubkowski,A.Wlodawer

Key ref:

J.Lubkowski et al. (2020). Mechanism of Catalysis by l-Asparaginase. Biochemistry, 59, 1927-1945. PubMed id: 32364696 DOI: 10.1021/acs.biochem.0c00116

Date:

22-Nov-19 Release date: 20-May-20

Protein chains

P00805  (ASPG2_ECOLI) -  L-asparaginase 2 from Escherichia coli (strain K12)

Seq: 348 a.a.

Struc: 327 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

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

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

Added reference

DOI no: 10.1021/acs.biochem.0c00116

Biochemistry 59:1927-1945 (2020)

PubMed id: 32364696

Mechanism of Catalysis by l-Asparaginase.

J.Lubkowski, J.Vanegas, W.K.Chan, P.L.Lorenzi, J.N.Weinstein, S.Sukharev, D.Fushman, S.Rempe, A.Anishkin, A.Wlodawer.

ABSTRACT

Two bacterial type II l-asparaginases, from Escherichia coli and Dickeya chrysanthemi, have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lymphomas. Despite a long history of successful pharmacological applications and the apparent simplicity of the catalytic reaction, controversies still exist regarding major steps of the mechanism. In this report, we provide a detailed description of the reaction catalyzed by E. coli type II l-asparaginase (EcAII). Our model was developed on the basis of new structural and biochemical experiments combined with previously published data. The proposed mechanism is supported by quantum chemistry calculations based on density functional theory. We provide strong evidence that EcAII catalyzes the reaction according to the double-displacement (ping-pong) mechanism, with formation of a covalent intermediate. Several steps of catalysis by EcAII are unique when compared to reactions catalyzed by other known hydrolytic enzymes. Here, the reaction is initiated by a weak nucleophile, threonine, without direct assistance of a general base, although a distant general base is identified. Furthermore, tetrahedral intermediates formed during the catalytic process are stabilized by a never previously described motif. Although the scheme of the catalytic mechanism was developed only on the basis of data obtained from EcAII and its variants, this novel mechanism of enzymatic hydrolysis could potentially apply to most (and possibly all) l-asparaginases.