TABLE 3.
Binding affinity of wild-type Ape1 and its mutant variants for insoluble O-acetyl-PG
Enzymes (20 μg) were incubated on ice with 200 μg of insoluble O-acetyl-PG suspended in 50 mm sodium phosphate buffer, pH 6.5, for 1 h prior to recovery of enzyme-bound ligand by ultracentrifugation.
| Enzyme | % Bounda | % Changeb |
|---|---|---|
| Ape1a wild type | 45.6 | |
| Catalytic triad | ||
| S80A | 1.64 | −96.4 |
| D366A | 14.1 | −69.1 |
| H369A | 0 | −100 |
| Potential binding residues | ||
| G78A | 0 | −100 |
| D79A | 0 | −100 |
| H81F | 27.7 | −39.3 |
| N235A | 73.1 | +160 |
| G236A | 37.4 | −18.0 |
| T267A | 0 | −100 |
| N268L | 1.95 | −95.7 |
| N268Q | 75.5 | +166 |
| N268S | 12.2 | −73.2 |
| V368A | 68.6 | +150 |
a Representative data calculated based on the amount remaining in the supernatant following incubation with ligand.
b % Change in PG binding ability of mutant derivatives relative to wild-type enzyme.