Figure 2.

(A) stopped-flow UV-vis absorbance traces of (16, 21 and 31 μM) wild-type thioesterase-catalyzed hydrolysis of (112 μM) 4-HB-CoA carried out under multiple turnover conditions and monitored at 300 nm (Δε = 11.8 mM⁻¹ cm⁻¹). The traces were fitted using a double exponential equation. (B) The time course for the multiple turnover reaction of 50 μM [¹⁴C]4 HB-CoA and 10 μM thioesterase monitored using rapid quench/HPLC techniques. The curves were simulated using the kinetic model shown in Scheme 2 and the microscopic rate constants k₁ = 172 μM⁻¹ s⁻¹, k₋₁ = 30 s⁻¹, k₂ = 264 s⁻¹, k₋₂ = 9 s⁻¹, k₃ = 25 s⁻¹, k₋₃ = 0 s⁻¹, k₄ = 129 s⁻¹ , k₋₄ = 5 μM⁻¹ s⁻¹, k₅ = 368 μM⁻¹ s⁻¹ and k₋₅ = 5 μM⁻¹ s⁻¹. The simulated curve-coloring scheme is as follows: E-I(CoA) + E(4-HB)(CoA) + E(4-HB) + 4-HB (red); E(4-HB-CoA) + 4-HB-CoA (black); E-I(CoA) (green); E(4-HB)(CoA) + E(4-HB) + 4-HB (blue).