FIGURE 5.

Dashed lines are the simulated transient population of various oxamate forms after T-jump from 20°C to 35°C using the parameters for the three-step kinetic model given in Eq. 1 (McClendon et al. (2)). The total oxamate and LDH/NADH concentrations were 0.6:0.6 mM. The transient spectra start from the equilibrium concentrations of various species containing oxamate at 20°C and then the 35°C parameters for the kinetic model are used to simulate the time course of each species. The solid curves show the single or biexponential fitting for each simulated time-dependent concentration. The fitted rate constants are also indicated. For comparison to the experimental data of Fig. 3, the simulated kinetics for LDH³/NADH·oxamate should be compared to the kinetic results taken at 1606 cm⁻¹, the marker band of the major species, whereas that for LDH²/NADH·oxamate would be compared to the results taken at 1624 cm⁻¹, the marker band for the minor species. The simulated relaxation kinetics for the major species show only one relaxation rate of 400 s⁻¹, in clear contrast to the two rates at 4200 s⁻¹ and 700 s⁻¹, respectively, observed in our experiments (see Fig. 3). The simulated relaxation for the minor species shows one rate at 8000 s⁻¹, not consistent with the observed rate at 300 s⁻¹. Furthermore, the populations of both major and minor species change in the same direction, also not consistent with our observations (see Fig. 3).