Analysis of PUGNAc and NAG-thiazoline as Transition State Analogues for Human O-GlcNAcase: Mechanistic and Structural Insights into Inhibitor Selectivity and Transition State Poise
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- 作者：Garrett E. Whitworth ; Matthew S. Macauley ; Keith A. Stubbs ; Rebecca J. Dennis ; Edward J. Taylor ; Gideon J. Davies ; Ian R. Greig ; David J. Vocadlo
- 刊名：Journal of the American Chemical Society
- 出版年：2007
- 出版时间：January 24, 2007
- 年：2007
- 卷：129
- 期：3
- 页码：635 - 644
- 全文大小：258K
- 年卷期：v.129,no.3(January 24, 2007)
- ISSN：1520-5126
文摘
O-GlcNAcase catalyzes the cleavage of 
s/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-O-linked 2-acetamido-2-deoxy-s/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-D-glucopyranoside(O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Two potent inhibitorsof this enzyme are O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc)and 1,2-dideoxy-2'-methyl-s/gifchars/alpha.gif" BORDER=0>-D-glucopyranoso[2,1-d]-s/gifchars/Delta.gif" BORDER=0 >2'-thiazoline (NAG-thiazoline). Derivatives of theseinhibitors differ in their selectivity for human O-GlcNAcase over the functionally related human lysosomals/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-hexosamindases, with PUGNAc derivatives showing modest selectivities and NAG-thiazoline derivativesshowing high selectivities. The molecular basis for this difference in selectivities is addressed as is howwell these inhibitors mimic the O-GlcNAcase-stabilized transition state (TS). Using a series of substrates,ground state (GS) inhibitors, and transition state mimics having analogous structural variations, we describelinear free energy relationships of log(KM/kcat) versus log(KI) for PUGNAc and NAG-thiazoline. Theserelationships suggest that PUGNAc is a poor transition state analogue, while NAG-thiazoline is revealedas a transition state mimic. Comparative X-ray crystallographic analyses of enzyme-inhibitor complexesreveal subtle molecular differences accounting for the differences in selectivities between these two inhibitorsand illustrate key molecular interactions. Computational modeling of species along the reaction coordinate,as well as PUGNAc and NAG-thiazoline, provide insight into the features of NAG-thiazoline that resemblethe transition state and reveal where PUGNAc fails to capture significant binding energy. These studiesalso point to late transition state poise for the O-GlcNAcase catalyzed reaction with significant nucleophilicparticipation and little involvement of the leaving group. The potency of NAG-thiazoline, its transition statemimicry, and its lack of traditional transition state-like design features suggest that potent rationally designedglycosidase inhibitors can be developed that exploit variation in transition state poise.