Jump to main content
Jump to site search

Issue 47, 2017, Issue in Progress
Previous Article Next Article

Computational insights into substrate binding and catalytic mechanism of the glutaminase domain of glucosamine-6-phosphate synthase (GlmS)

Author affiliations

Abstract

Glucosamine-6-phosphate synthase (GlmS) is a key enzyme in the biosynthesis of hexosamine across a variety of species including Escherichia coli, fungi, and humans. In particular, its glutaminase domain catalyzes the conversion of glutamine to glutamic acid with the release of ammonia. A catalytically important cysteinyl (Cys1) has been suggested to act as the mechanistic nucleophile after being activated by the N-terminal amine of the glutaminase domain (i.e., its own α-amine). Using molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) computational methods, we have investigated the active site of the glutaminase domain, the protonation state of its N-terminal amine, substrate binding, and catalytic mechanism. In addition, the potential for an active site histidyl (His71) to alternatively act as the required base was examined. The N-terminal amine is concluded to have a reduced pKa due to being buried within the enzyme and the nearby presence of a protonated arginyl residue. Previous suggestions that this was due in part to hydrogen bonding with the hydroxyl of Thr606 is not supported; such an interaction is not consistent, and accounts for only 4% of the total duration of the MD simulation. The most feasible enzymatic pathway is found to involve a neutral N-terminal Cys1 α-amine acting as a base and directly deprotonating (i.e., without the involvement of a water, the Cys1SH thiol). The tetrahedral oxyanion intermediate formed during the mechanism is stabilized by a water and two enzyme residues: Asn98 and Gly99. Furthermore, the overall rate-limiting step of the mechanism is the nucleophilic attack of a water on the thioester cross-linked intermediate with a barrier of 74.4 kJ mol−1. An alternate mechanism in which His71 acts as the nucleophile-activating base, and which requires the Cys1 α-amine to be protonated, is calculated to be enzymatically feasible but to have a much higher overall rate-limiting barrier of 93.7 kJ mol−1.

Graphical abstract: Computational insights into substrate binding and catalytic mechanism of the glutaminase domain of glucosamine-6-phosphate synthase (GlmS)

Back to tab navigation

Supplementary files

Publication details

The article was received on 02 May 2017, accepted on 02 Jun 2017 and first published on 07 Jun 2017


Article type: Paper
DOI: 10.1039/C7RA04906D
Citation: RSC Adv., 2017,7, 29626-29638
  • Open access: Creative Commons BY-NC license
  •   Request permissions

    Computational insights into substrate binding and catalytic mechanism of the glutaminase domain of glucosamine-6-phosphate synthase (GlmS)

    W. Wei, G. Monard and James W. Gauld, RSC Adv., 2017, 7, 29626
    DOI: 10.1039/C7RA04906D

    This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material and it is not used for commercial purposes.

    Reproduced material should be attributed as follows:

    • For reproduction of material from NJC:
      [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the RSC.
    • For reproduction of material from PCCP:
      [Original citation] - Published by the PCCP Owner Societies.
    • For reproduction of material from PPS:
      [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC.
    • For reproduction of material from all other RSC journals:
      [Original citation] - Published by The Royal Society of Chemistry.

    Information about reproducing material from RSC articles with different licences is available on our Permission Requests page.

Search articles by author

Spotlight

Advertisements