Identical amounts of proteins (600 g) were transferred to Eppendorf tubes, and 40 g of Streptavidin agarose beads was added to isolate biotin-tagged plasma membrane GLUT4. sensing,2regulation of gene expression,3target protein activity modification4and protein degradation.5Disregulation ofO-GlcNAc modification has been implicated in insulin resistance6and Alzheimers disease.7The recent discovery that transcription factor Forkhead Box Fox01 is regulated by thisO-GlcNAc modification provides a potential mechanism by which hyperglycemia promotes T-1095 gluconeogenesis and worsening of glucose intolerance.8O-GlcNAc metabolism is usually regulated by two key enzymes:O-GlcNAc transferase (OGT) andO-GlcNAcase (OGA). OGT catalyzes the transfer ofO-GlcNAc from a UDP-GlcNAc donor to the various target proteins, and OGA catalyzesO-GlcNAc hydrolytic removal.9A number of mechanistic and inhibition studies10,11have demonstrated that two aspartic acids (Asp174 and 175) in the OGA active site of the N-terminal domain are key catalytic residues that enable a substrate-assisted mechanism. The Rabbit Polyclonal to Chk2 (phospho-Thr387) inhibitor GlcNAc-thiazoline (1) and its relatives bind in enzyme active sites in the pseudo-chair (4C1) conformation. Based on the correlation of free energy of activation calculated from enzymatic substrate hydrolysis relative to G for inhibitor binding, Vocadlo and co-workers concluded that1and its analogues behave specifically as OGA transition state mimics.11In this paper, we evaluate comparative OGA inhibition by the GlcNAc-selenazoline (2), a close analogue of1, but with greater lipophilicity and longer carbonchalcogen bonds. OGA uses a double displacement mechanism (Fig. 1) that features substrate-assisted catalysis: the substrateAbinds in the1S3twist vessel conformation with co-linear orientation of the leaving aglycon T-1095 oxygen and the approaching amide carbonyl oxygen. Bond rupture at the anomeric carbon (C-1) is usually accompanied by participation of the amide carbonyl from the opposite face. During the intramolecular SN2-like reaction, the pyranose ring flattens about C-1, and then inverts toward T-1095 a bicyclic oxazolinium intermediateCthrough transition stateB. On the basis of studies that include linear free energy correlations, X-ray crystallographic analyses, and computational modeling, Vocadlo and co-workers suggest that the thiazoline1in the chair conformation is usually closer to the trigonal bipyramidal SN2 transition stateB(Fig. 1) rather than the T-1095 oxazolinium intermediateC.11The highly structurally analogous -Glc-NAc-selenazoline2might be anticipated to inhibit OGA analogously. == Physique 1. == Substrate-assisted catalytic hydrolysis mechanism used by OGA (A [B] C D). == 2. Results and discussion == == 2.1. Synthesis of GlcNAc-selenazoline == We have prepared2(Scheme 1) by treating -d-glucosamine penta-acetate4with the Woollins reagent, [PhP(=Se)Se]2,12followed by O-deacetylation. The purpose of the pyridine in the selenonation reaction is usually to neutralize the acid that accumulates during the cyclization. Without the added base, the acetic acid liberated by product formation is usually converted13by the Woollins reagent to the (more acidic) selenoacetic acid, which causes counterproductive O-cyclization of4to an oxazoline. == Scheme 1. == == 2.2. OGA inhibition by GlcNAc-selenazoline == Evaluation of OGA inhibition by2was carried out by using FDGlcNAc (3)14as the OGA substrate. A LineweaverBurk plot of the initial velocity at various concentrations of2shows that2competitively inhibits OGA with aKiof 0.7 M (Fig. 2). Compared with1(Ki= 0.01 M),2is obviously a much less potent competitive inhibitor (about 70-fold; seeFig. 3). For comparison, competitive inhibitionKis for1and2against a hexosaminidase from the bacteriumStreptomyces plicatus(withp-nitrophenyl -d-N-acetylglucosaminide as the substrate)15are 20 and 170 M, respectively, an eightfold difference. The disparity between1and2as inhibitors might be attributable to, among other factors, the longer carbonchalcogen bond distance in2, and the poorer ability of the divalent selenium atom (relative to sulfur) to act as a hydrogen bond acceptor in the active site. AmongN-acetylhexosaminidases, OGA is known to be relatively tolerant of small steric differences in the neighborhood of the substrate acetamido methyl group,11,16but in this instance is usually responding dramatically to changes at the anomeric participating heteroatom. == Physique 2. == LineweaverBurk analysis of OGA inhibition in the absence and the presence of increasing concentrations of2with FDGlcNAc3as substrate. == Physique 3. == LineweaverBurk analysis of OGA inhibition in the absence and the presence of increasing concentrations of1with FDGlcNAc3as substrate. The4C1chair conformation of2(shown) is usually calculated to be the most stable, according to ab initio calculations17(B3LYP/6-31G*), which also estimate respective CSe bond lengths of 2.03 (to C-1) and 1.94 . Two other conformations,0S2and1S3, are energetically accessible and may also contribute in answer. By comparison, small molecule crystallographic data for the4C1conformation of a 6-O-acylated derivative of1indicate respective C-S distances of 1 1.85 (to C-1) and 1.76 , and the corresponding calculated (B3LYP/6-31G*) distances are 1.89 and 1.80 , respectively.17The respective carbonchalcogencarbon bond angles of1and2are calculated to be 87.8 and 83.2 (4C1conformations), whereas the crystallographic CSC angle is 88.8. The calculated4C1conformations of the GlcNAc-oxazoline, -thiazoline (1), and – selenazoline (2) are displayed inFigure 4, overlapped at C-1 and C-2 so as to highlight the differences around the chalcogen and anomeric carbon. == Physique 4. == Calculated4C1structures of oxazoline intermediate C, thiazoline1, and selenazoline2overlapped at C-1 and C-2 (carbohydrate numbering), showing the respective chalcogens.
