Table 1.
Hydrophobic Stacking Interactions of Tyr69 Are Essential for Substrate Binding by hOGA
| TAB1 Peptide |
hOGA Peptide |
p53 Peptide |
p53 Peptide |
|
|---|---|---|---|---|
| PVSVPYS(O-GlcNAc)SAQSTS | VAHS(O-GlcNAc)GAK | VDS(O-GlcNAc)TPG | QLWVDS(O-GlcNAc)TPPPG | |
| Y69S | >4,000 | >4,000 | 3,400 ± 500 | 290 ± 20 |
| Y69K | >4,000 | >4,000 | >4,000 | 350 ± 20 |
| Y69Q | 3,600 ± 300 | >4,000 | >4,000 | 170 ± 40 |
| Y69F | 1,100 ± 60 | 1,200 ± 110 | 2,700 ± 400 | 53 ± 5 |
| Wild-type | 940 ± 80 | 790 ± 40a | 1,300 | 21 ± 3 |
Tyr69 in human OGA may participate in hydrogen bonding with the catalytic aspartate (Asp175) as well as providing a stacking platform for the −1/−2 peptide bonds in the substrate. The Y69S mutation disrupts both these interactions, the Y69K and Y69Q mutants cannot participate in pi-pi stacking, and Y69F abolishes the hydrogen bond. Point mutations were introduced in hOGA, and the Michaelis constant (KM, given in μM) was determined for several peptide substrates.