R represents a CH₃ group in MMSA and PPCoA, whereas it represents a hydrogen atom in MSA and acetyl-CoA.

Assays were performed at 30 °C under the following conditions: 50 mM potassium phosphate buffer (pH 8.2), 2 mM NAD, 500 μM MMSA and 500 μM CoA. In curve A, the reaction was initiated by adding 0.125 μM apo-MSDH and the steady-state rate was attained after approx. 5 min (solid line). In curve B, the enzyme pre-incubated for 20 min in the presence of 2 mM NAD was added to the complete assay to initiate the reaction.

Fluorescence quenching was recorded after addition of 2 mM NAD to 1.25 μM wild-type (WT) and W468F MSDHs dissolved in 50 mM potassium phosphate buffer, pH 8.2 at 30 °C. The inset details the fluorescence quenching recorded for the W28F, W76F, W177F and W397F MSDHs under the same experimental conditions. Excitation and emission wavelengths were 297 and 335 nm respectively. The raw fluorescence quenching data were converted into relative fluorescence intensities using the fluorescence intensity of the apo-forms as a reference.

Apo-(wild-type) MSDH (1.25 μM) was incubated in the presence of NAD (0.25–5 mM) in 50 mM potassium phosphate buffer, pH 8.2 at 30 °C. The time-course of the MSDH fluorescence change induced by NAD-binding was then recorded at 335 nm. At each NAD concentration a k_obs value was calculated by fitting the slow additional fluorescence quenching to a mono-exponential equation. Data were fitted to eqn (1) demonstrating a K_app of 0.38±0.08 mM and a k_{obs max} of 0.18±0.01 min⁻¹.

Kinetic studies were performed over a pH range of 5.5–9.0 or 6.0–9.0 in buffer A at 30 °C. The MSDH concentration was 1.55 μM and 2PDS was 20- or 30-fold in excess relative to the free-SH concentration. Experimental data were analysed as described by Marchal and Branlant [8]. (A) Transients obtained on the apo-form were best described by a double exponential expression reflecting the contribution of two populations of non-equivalent Cys³⁰² present in equal concentrations (at pH≤6.5, only one population was observed). Both data sets were fitted against a one-pKa model, which gave a pK_app of approx. 8.7 but different k₂ values of 1.4×10⁵ M⁻¹·s⁻¹ (●) and 1.8×10⁴ M⁻¹·s⁻¹ (◆) respectively. (B) As described for the apo-form, two populations of Cys³⁰² were also characterized in the holo-form. Data sets were fitted against a one-pKa model, which gave k₂ and pK_app values of 77 M⁻¹·s⁻¹ and 7.90 (■), and 5.1×10⁴ M⁻¹·s⁻¹ and 8.60 (▲) respectively. As described above, the two Cys³⁰² populations were not distinguishable at pH≤6.5. Thus the k₂ value of approx. 20 M⁻¹·s⁻¹ exhibited by the ‘activated’ Cys³⁰² (■) at pH≤6.5 reflects the contribution of the ‘non-activated’ population (▲) (k₂ approx. 130 M⁻¹·s⁻¹ at pH 6.0). Therefore the pK_app value of 7.9 is slightly overestimated.

The experiment was carried out under pre-steady-state conditions in 50 mM potassium phos-phate buffer (pH 8.2) at 30 °C and in the absence of CoA. Under these experimental conditions, accumulation of the thioacylenzyme is observed due to the very low steady-state rate, 10⁻³·s⁻¹. After mixing, the reaction contained 2 mM NAD, 500 μM MMSA and 4 μM (curve A) or 8 μM (curve B) of ‘activated’-MSDH. The burst of NADH production was monitered at 340 nm. The extrapolated intercept from the steady-state rate portion corresponds to a burst of 0.5 mol of NADH/mol of MSDH monomer.

Pre-steady-state data were collected at 10 °C on a SX18MV-R stopped-flow apparatus (Applied PhotoPhysics) using the 5 μl cell, by rapidly mixing 4 μM wild-type MSDH, 2 mM NAD and 10 mM MMSA (final concentrations) over a pH-range of 5–8.5 in buffer A. At pHs lower than 5, MSDH was not stable. NADH appearance was monitored at 340 nm and experimental data (●) were best fitted by non-linear regression analysis against a one-pKa model, identified by the best-fit theoretical curve (solid line).

(A) A solution of 7.5 μM LDH, 20 mM pyruvate and 1 mM MMSA was rapidly mixed with an equal volume of 7.5 μM ‘activated’-MSDH and 4 mM NAD. Both syringes contained 50 mM potassium phosphate buffer, pH 8.2. Under the same experimental conditions, the rate of oxidation of free NADH by LDH was shown to be 130 s⁻¹. Data collected were best fitted to a triphasic expression using the Origin 7 software (Microcal), as shown by the lack of systematic deviation in the plot of residuals (lower panel). The first phase represents the acylation step, the second one the NADH consumption after its release from the thioacylenzyme–NADH complex and the third one could be due to the reverse LDH-catalysed reaction. The rate constants obtained for the global fitting are 72±1 s⁻¹, 56±1 s⁻¹, and (3.00±0.07)×10⁻² s⁻¹ respectively. (B) The rate of the decarboxylation process was determinated at 30 °C from the thioacylenzyme intermediate by using the PEP/PEPC/MDH system as a coupled assay. Experimental conditions are described in detail in the Experimental section. Data collected using a split time-base (2000 points for the first 100 ms, and 2000 points for the remaining 10 s) were best fitted to a triphasic expression using the Origin 7 software as shown by the lack of systematic deviation in the plot of residuals (lower panel). The first phase represents the acylation step, the second one the bicarbonate release and the third one could be due to the reverse MDH-catalysed reaction. The rate constants obtained for the global fitting are 415±5 s⁻¹, 2.5±0.1 s⁻¹, and (1.00±0.01)×10⁻³ s⁻¹ respectively.