TABLE 2.
Glycosylation turnover rates and dissociation constants for PglP and peptide variants affecting interactions at the +2 position
Turnover rates and dissociation constants were derived from curves shown in supplemental Figs. S2 and S4. The errors represent the S.E. of the fit for each turnover rate and dissociation constant. Values for relative turnover and relative binding refer to the ratio of an individual mutant/sequon and WT PglB/DQNAT sequon.
| Mutant | Sequon | Turnover rate | Relative turnover/fold reduction | Dissociation constant (Kd) | Relative binding |
|---|---|---|---|---|---|
| peptides/s | μm | ||||
| WT | DQNAT | 1.50 ± 0.04 | 1 | 1.02 ± 0.06 | 1 |
| DQNAS | 1.25 ± 0.04 | 0.83/1.2-fold | 4.02 ± 0.20 | 0.25 | |
| DQNAC | (3.65 ± 0.12) × 10−3 | 2.43 × 10−3/400-fold | –a | ||
| DQNAV | (2.21 ± 0.07) × 10−4 | 1.47 × 10−4/7,000-fold | –a | ||
| DQNAA | (3.60 ± 0.12) × 10−4 | 2.40 × 10−4/4,000-fold | –a | ||
| W464F | DQNAT | (1.55 ± 0.05) × 10−3 | 1.03 × 10−3/1,000-fold | –a | |
| I572V | DQNAT | 1.08 ± 0.02 | 0.72/1.4-fold | 3.75 ± 0.15 | 0.27 |
| DQNAS | 0.61 ± 0.02 | 0.41/2.5-fold | 7.71 ± 0.11 | 0.13 | |
| DQNAA | (3.60 ± 0.06) × 10−4 | 2.40 × 10−4/4,000-fold | NDb | ||
| I572A | DQNAT | 0.33 ± 0.08 | 0.22/4.5-fold | 57.31 ± 0.93 | 0.02 |
| DQNAS | 0.13 ± 0.04 | 8.67 × 10−2/12-fold | 165.60 ± 5.50 | 0.01 | |
| DQNAA | (1.14 ± 0.05) × 10−4 | 7.60 × 10−5/13,000-fold | ND |
a Peptide binding was observed, but no Kd could be determined because the fluorescence anisotropy signal reached only 30–50% saturation at the limiting PglB concentration of 100–150 μm.
b ND, data not determined.