Molecular modeling insights into the inhibition and catalytic mechanism of Golgi a-Mannosidase II
Kawatkar, Sameer Pralhad
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In this dissertation, we have employed a computational methodology in conjunction with experimental techniques to understand the mechanism of inhibition of Golgi a-Mannosidase II (GMI) by mannostatin A, and to determine the mechanism of the hydrolysis reaction catalyzed by GMII. Ab initio calculations and molecular docking studies were employed to rationalize the inhibition data of mannostatin A analogues and to identify the mode of binding of mannostatin A. It was found that Mannostatin and aminocyclopentitretrol could bind to GMII in a similar mode as that of the known inhibitor Swainsonine. However, due to the flexibility of the five membered rings of these compounds, additional low energy binding modes could be adopted. The thiomethyl moiety of mannostatin was predicted to make favorable hydrophobic interactions with Arg228 and Tyr727, possibly accounting for its greater inhibitory activity. In order to validate the docking predictions, the X-ray crystal structures of Drosophila Golgi ±-mannosidase II (dGMII) complexed with the inhibitors mannostatin A and its N-benzyl analog have been determined. The X-ray structures were in excellent agreement with the predicted binding modes for mannostatin A analogues. Molecular dynamics simulations and NMR studies have shown that the five-membered ring of mannostatin A is rather flexible. In the 2bound state, mannostatin A adopts a T1 twist envelope conformation, which is not significantly populated in solution. Possible conformations of the mannosyl oxacarbenium ion and an enzyme-linked intermediate have been compared to the conformation of mannostatin A in the co-crystal structure with dGMII. It has been found that mannostatin A best mimics the covalent linked mannosyl intermediate. The thiomethyl group is able to make a number of additional polar and non-polar interactions increasing the affinity for dGMII. Finally, a quantum mechanical investigation of the catalytic mechanism of the hydrolysis reaction catalyzed by GMII has been conducted. These calculations have revealed that in the transition state, the mannopyranosyl ring adopts a conformation that lies between an ideal 1B2,5 and S5 conformation. Superimposition of mannostatin A and swainsonine in their bound conformations onto the optimized transition state structure revealed that both inhibitors mimic the transition state.