Enzyme

6.3.5.4 - Asparagine synthase (glutamine-hydrolyzing)

Alternative Name(s)
  • Asparagine synthetase (glutamine-hydrolyzing).
  • Asparagine synthetase B.
  • Glutamine-dependent asparagine synthetase.
  • AS-B.

Catalytic Activity

ATP + H2O + L-aspartate + L-glutamine = AMP + diphosphate + H(+) + L-asparagine + L-glutamate

Cofactors

There are no Cofactors for this Enzyme

Reaction Mechanism

    Asparagine synthetase B catalyses the ATP dependent formation of asparagine from aspartate using either glutamine or ammonia as a nitrogen source. The enzyme is a homodimer with two active sites [PMID:10587437] in each subunit, one responsible for glutamine hydrolysis and one for asparagine synthesis, separated by a hydrophobic intramolecular tunnel around 20A long [PMID:12706338]. Sequencing analysis has shown the synthetase to belong to the larger glutamine transferase family.

    The enzyme catalyses three distinct chemical reactions: glutamine hydrolysis to yield ammonia (which is then channelled to the second active site) takes place in the N-terminal domain. The C-terminal active site mediates both the synthesis of a beta-aspartyl-AMP intermediate and its subsequent reaction with ammonia. However, the exact order of the partial reactions is still somewhat unclear [PMID:9748330, PMID:12706338], here we show the hydrolysis of the glutamate occurring first.

    Glutamine hydrolysis occurs at the N terminal. A nucleophilic Cys residue attacks the glutamine substrate, displacing ammonia, forming a substrate-enzyme intermediate which is then hydrolysed. The ammonia diffuses though the interdomain tunnel from the site of production to the site of utilisation: the synthetase component in the C terminal.

    A two site ping pong mechanism for the reaction between the aspartic acid, ATP and ammonia has been implicated with several residues thought to be responsible for the binding the substrates through hydrogen bonding interactions.

    Kinetic analysis has shown the rate of catalysis at each site to be independent of one another, and so the stoichiometry between the sites must be maintained by the catalytic efficiency of the two sites.

    Catalytic Residues
    AA Uniprot Uniprot Resid PDB PDB Resid
    Cys P22106 2 1ct9 1
    Thr P22106 322 1ct9 321
    Leu P22106 51 1ct9 50
    Gly P22106 76 1ct9 75
    Arg P22106 325 1ct9 324
    Cys P22106 2 1ct9 1
    Asn P22106 75 1ct9 74
    Step Components

    overall reactant used, proton transfer, unimolecular elimination by the conjugate base, overall product formed, enzyme-substrate complex formation, bimolecular nucleophilic addition, intermediate formation, native state of enzyme regenerated, proton relay, deamination, intermediate collapse, enzyme-substrate complex cleavage

    Step 1.

    This reaction takes place in the glutaminase domain. The N-terminus of Cys1 deprotonates water, which deprotonates the thiol group of Cys1, initiating a nucleophilic attack on the amide carbon in an addition reaction.

    Step 2.

    This reaction takes place in the glutaminase domain. The oxyanion initiates an elimination that cleaves ammonia from the bound L-glutamine substrate. Ammonia deprotonates water, which deprotonates the N-terminus of Cys1. The ammonia product is then transferred to the second domain of the enzyme through an internal ion channel

    Step 3.

    This reaction occurs in the glutaminase domain. The N-terminus of Cys1 deprotonates water, which initiates a nucleophilic attack on the carbonyl carbon of the covalently bound intermediate in an addition reaction.

    Step 4.

    This reaction occurs in the glutaminase domain. The oxyanion initiates an elimination that cleaves the C-S bond, the thiolate of Cys1 deprotonates water, which deprotonates the N-terminus of Cys1.

    Step 5.

    This reaction occurs in the ligase domain of the enzyme. The aspartate substrate initiates a nucleophilic attack on the alpha phosphate of ATP in an addition reaction.

    Step 6.

    This reaction occurs in the ligase domain of the enzyme. The pentavalent intermediate collapses to eliminate the diphosphate product and the beta-Asp-AMP intermediate.

    Step 7.

    This reaction occurs in the ligase domain of the enzyme. The ammonia nitrogen initiates a nucleophilic attack on the carbonyl carbon of the beta-Asp-AMP intermediate in an addition reaction.

    Step 8.

    This reaction occurs in the ligase domain of the enzyme. The oxyanion initiates an elimination that cleaves the C-O bond, the phosphate of AMP deprotonates the ammonium group, releasing the AMP and Asparagine products.

    Products.

    The products of the reaction.

Reaction Parameters

There are no kinetic parameters information for this Enzyme

Associated Proteins

Protein name Organism
Probable asparagine synthetase [glutamine-hydrolyzing] Fission yeast
Asparagine synthetase [glutamine-hydrolyzing] 2 Mouse-ear cress
Asparagine synthetase [glutamine-hydrolyzing] 3 Mouse-ear cress
Putative asparagine synthetase [glutamine-hydrolyzing] Mycobacterium bovis (strain ATCC BAA-935 / AF2122/97)
Asparagine synthetase [glutamine-hydrolyzing] 1 Mouse-ear cress

Citations