Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects -- Hay et al. 104 (2): 507 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Hay et al. 10.1073/pnas.0608408104.

Supporting Figures

Files in this Data Supplement:

SI Figure 5
SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9

SI Figure 5

Fig. 5. Deconvoluted absorbance spectra from the reductive half-reaction of 20 mM MR mixed with 50 mM NADH at 10°C. The spectra were deconvoluted in an irreversible two-step reaction (a®b®c). (A and B) Spectra for the fully-oxidized enzyme (A) and the CT complex (B) are shown versus pressure. (C) The reduced enzyme is essentially colorless and not shown. The first derivative spectra (de/dl) are also shown (´10 and baseline offset) to illustrate that there is absolutely no detectable shift in the spectra of either A or B with pressure. (C) Absorption spectra of MR in the absence (native) and presence of 10 M urea (denatured) showing the ≈15-nm shift of the 464-nm peak upon denaturation of the enzyme. (D) The pressure dependence of the magnitude of the charge-transfer absorbance at 552 nm. These values are the average of four separate experiments and have been normalized. e is the molar extinction coefficient.

SI Figure 6

Fig. 6. The dependence of the observed rate for the reductive half-reaction of MR on NADH concentration at 10°C and 40°C. The apparent K_d values at 1 bar, 1 kbar, and 2 kbar were determined by fitting the data to k_obs = k_max [NADH]/(K_d [NADH])

SI Figure 7

Fig. 7. The effect of temperature on DV^‡ for the reductive half-reaction of MR with saturating NADH (black) and NAD²H (red). The difference in DV^‡ is shown in blue below. The solid lines are linear fits and the dotted black line through the DDV^‡ data are a fit omitting the data point at 40°C (313 K).

SI Figure 8

Fig. 8. The effect of pressure on the enthalpy (DH^‡) (Top) and entropy (DS^‡) (Middle) of the reductive half-reaction of MR with saturating NADH (black) and NAD²H (red). The difference in DH^‡ and DS^‡ between the reaction with NAD²H and NADH are shown in blue. (Bottom) The lnA' values and prefactor ratio (A'_H :A'_D, blue) are shown.

SI Figure 9

Fig. 9. Additional numerical modeling. (A) The effect of r₀ on the harmonic oscillator force constant required for KIE = 4.0. (B) The apparent difference in enthalpy when the KIE = 4.0. (C) The frequency of gating for a range of gating unit masses (atomic mass units) when the difference in enthalphy (DDH^‡) is fixed to 5.2 kJ·mol^-1. (D) The Eyring prefactor ratio when the difference in enthalpy (DDH^‡) is fixed to 5.2 kJ·mol^-1.