Table 2. Pre-steady state kinetic parameters of T4 Dam MTase interaction with duplexes containing a native or modified recognition site at limiting enzyme concentrationa.
Oligonucleotide substrate | Combination of duplexes | Recognition sequenceb | Burstc | kmeth (s–1)c | kcat (s–1)c |
---|---|---|---|---|---|
Canonical sites | |||||
-G-A-T-C- | 0.92 | 0.56 | 0.023 | ||
1 | I + II | -C-T-A-G- | (0.05) | (0.10) | (0.002) |
-G-M-T-C- | 0.85 | 0.47 | 0.021 | ||
1m | Im + II | -C-T-A-G- | (0.05) | (0.09) | (0.002) |
-G-M-T-C- | 0.84 | 0.14 | 0.021 | ||
1md | Im + II | -C-T-A-G- | (0.07) | (0.04) | (0.002) |
Substituted sites | |||||
-G-A-T-C- | 0.86 | 0.49 | 0.0051 | ||
2 | I + III | -C-A-T-G- | (0.05) | (0.09) | (0.0005) |
-G-A-T-C- | 0.90 | 0.67 | 0.0038 | ||
3 | I + IIn | -C-T-N-G- | (0.03) | (0.12) | (0.0005) |
aEnzyme was preincubated with [3H-CH3]-AdoMet prior to the addition of duplex.
bN is 2-aminopurine; M is N6-methyladenine.
cValues in parentheses correspond to standard deviations.
dEnzyme was preincubated with duplex 1m before the addition of the [3H-CH3]-AdoMet.