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. Author manuscript; available in PMC: 2009 Jun 6.
Published in final edited form as: J Mol Biol. 2008 Apr 8;379(3):579–588. doi: 10.1016/j.jmb.2008.04.005

Table 1.

Kinetica parameters of CCA additionb.

Intact tRNA ASL-nicked tRNA Discrimination
kN (s-1) Kd (tRNA, μM) kN/Kd (s-1μM-1) kN (s-1) Kd (tRNA, μM) kN/Kd (s-1μM-1) kN/Kd (intact)/kN/Kd (nicked)
A76 170 ± 10 0.8 ± 0.2 212 170 ± 10 8.6 ± 1.3 20 10.6
C75 170 ± 20 2.8 ± 0.8 61 180 ± 20 17.5 ± 3.8 10 6.1
C74 170 ± 20 3.3 ± 0.8 51 150 ± 10 16.9 ± 4.2 8.9 5.7
a

The reported kinetic Kd (or Km(sto)) values represent the monomer concentration of EcCCA. In crystal structures of the class I AfCCA, the enzyme exists as a dimer and binds two molecules of tRNA or the minihelix domain.6; 7; 9; 11 In the crystal structure of the class II Bacillus stearothermophilus CCA enzyme, the enzyme also exists as a dimer and is modeled to bind two molecules of tRNA.5; 6

b

Values reported are based on experiments performed with pre-incubating EcCCA with a tRNA substrate in one syringe of the rapid quench instrument, followed by rapid mixing with NTP from the other syringe.