Table 2. Steady-state kinetic parameters of BamHI MTase methylation of variant duplexes containing a canonical or defective recognition site.
Substrate | ODN combination | Structure of recognition site | Km (nM) | kcat (× 10−3 s−1) | kcat/Km(M−1 s−1) | Relative kcat/Kma |
---|---|---|---|---|---|---|
d1 | n1 + n2 | –G–G–A–T–C–C– | 38.8 ± 4.2 | 17.7 ± 0.3 | 460 | (1.00) |
–C–C–T–A–G–G– | ||||||
d2 | n2 + n1m | –G–G–A–T–C–C– | 38.0 ± 4.0 | 16.0 ± 0.3 | 410 | 0.90 |
–C–C–T–M–G–G– | ||||||
d3 | n14b | –G–G–A–T–C–C– | 304 ± 53 | 9.2 ± 0.5 | 30 | 0.07 |
–C–C–T–A–G–G– | ||||||
d4 | n1 | –G–G–A–T–C–C– | 310 ± 43 | 5.8 ± 0.3 | 19 | 0.04 |
d5 | n1 + n13 | –G–G–A–T–C–C– | 1730 ± 390 | 0.58 ± 0.07 | 0.34 | 0.0007 |
–C–C–A–T–G–G– | ||||||
d6 | n1 + n3 + n4 | –G–G–A–T–C–C– | 95.6 ± 11.8 | 3.0 ± 0.1 | 31 | 0.07 |
–C∧C–T–A–G–G– | ||||||
d7 | n1 + n5 + n6 | –G–G–A–T–C–C– | 198 ± 46 | 0.14 ± 0.01 | 0.69 | 0.002 |
–C–C∧T–A–G–G– | ||||||
d8 | n1 + n7 + n8 | –G–G–A–T–C–C– | 274 ± 31 | 5.8 ± 0.2 | 21 | 0.05 |
–C–C–T∧A–G–G– | ||||||
d9 | n1 + n9 + n10 | –G–G–A–T–C–C– | 35.0 ± 3.7 | 14.0 ± 0.3 | 400 | 0.88 |
–C–C–T–A∧G–G– | ||||||
d10 | n1 + n11 + n12 | –G–G–A–T–C–C– | 18.2 ± 5.1 | 17.8 ± 0.8 | 980 | 2.2 |
–C–C–T–A–G∧G– | ||||||
d11 | n1 + n4 | –G–G–A–T–C–C– | 68.0 ± 11.0 | 2.3 ± 0.1 | 33 | 0.07 |
… C–T–A–G–G– | ||||||
d12 | n1 + n3 + n6 | –G–G–A–T–C–C– | — | 0 | — | — |
–C … T–A–G–G– | ||||||
d13 | n1 + n5 + n8 | –G–G–A–T–C–C– | — | 0 | — | — |
–C–C … A–G–G– | ||||||
d14 | n1 + n7 + n10 | –G–G–A–T–C–C– | 386 ± 100 | 0.43 ± 0.02 | 1.1 | 0.002 |
–C–C–T … G–G– | ||||||
d15 | n1 + n9 + n12 | –G–G–A–T–C–C– | 16.7 ± 2.9 | 12.5 ± 0.3 | 750 | 1.6 |
–C–C–T–A … G– | ||||||
d16 | n1 + n11 | –G–G–A–T–C–C– | 16.4 ± 4.4 | 22.0 ± 0.8 | 1300 | 3.0 |
–C–C–T–A–G … | ||||||
d17 | n1 + n9 | –G–G–A–T–C–C– | 11.8 ± 4.1 | 15.0 ± 1.3 | 1200 | 2.8 |
–C–C–T–A …… |
‘∧’Signifies the absence of an internucleotide phosphate; ‘…’ signifies absence of a nucleotide residue.
M = N6-methyladenine.
akcat/Km values are relative to that for canonical duplex 1.
b12mer self-complementary oligonucleotide.