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Michaelis-Menten Kinetics. K m is the substrate concentration at which the rate of the reaction is half the maximum rate (V max ). Transcription. 2-10 nM. 9-12 nt synthesis. 1x10 8 M -1 s -1. 30s -1. 150s -1. bent. F obs = F o (1- e -kt )+F b. Expected results. fluorescence. time.
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Michaelis-Menten Kinetics Km is the substrate concentration at which the rate of the reaction is half the maximum rate (Vmax)
Transcription 2-10 nM 9-12 nt synthesis 1x108 M-1s-1 30s-1 150s-1 bent
Expected results fluorescence time Measurement of the dissociation rate constant chase 2-AP High fluorescence No fluorescence excess No fluorescence low fluorescence 2-AP
Measurement of the dissociation rate constant 0.05 mM p-dsDNA-T7 RNAP complex (0.05 mM T7 RNAP + 0.1 mM -4T 2-AP p-dsDNA) with 2 mM non-fluorescent p-dsDNA in a standard fluorescence cuvette. Time-dependent fluorescence decrease at 370 nm (upon excitation at 315 nm) was monitored (circles). The fluorescence decrease was fit to Eqn. 6 that provided koff of 0.00034 s-1 (solid line). Thus the overall Kd (= koff/kon) of T7 RNAP-p-dsDNA complex is 1.3 pM.
kcat k1 k-1 Kd = k-1/k1
P1 E + S ES EI E+P2 P1 EI P2
P1 E + S ES EI E+P2 P1 EI P2
Observed rate = k1x[A] = 4 x 106 M-1s-1 x 100 x 10-6 M = 400 s-1 = 20 s-1
100 mmol/min 10 mM [S]
