Author ORCID Identifier : James W. Gauld

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RSC Advances



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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.

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