The structure of E. coli PGDH (pdb 1psd) is shown in ribbon diagram. Each of the subunits are colored to distinguish them in the figure. The positions of the domains are labeled. L-Serine (Pink) is shown in ball and stick form bound to two sites at each ACT domain interface (Green-Pink and Blue-Orange subunits). The position of E360 is shown in yellow ball and stick form in the ACT domains (top and bottom of figure). NADH (Green) and W139 (Blue) are shown in ball and stick form at each active site cleft.

The observed rates (k_obs) derived from the pre-steady state transients are plotted versus NADH concentration. k_obs 1(●), k_obs 2 (■), k_obs 3 (○), k_obs 4 (▲). The solid lines are fits of the data to equation 2. The protein concentration was 2.5 μM subunit.

The observed rates (k_obs) derived from the pre-steady state transients are plotted versus α-ketoglutarate concentration. The k_obs are determined in the presence of 0 (●), 2 (■), 3 (◆), and 4 μM (▲) L-serine. The solid line is a fit of the average of the values to equation 3. The protein concentration was 2 μM subunit and the NADH was 250 μM.

The quenching of tryptophan fluorescence as a function of L-Serine concentration is plotted. L-Serine was added in successive aliquots to 2.5 μM enzyme (subunits) solutions of W139F-E360W PGDH (■). The solid line is a fit of the data to equation 11.

The observed rates (k_obs) derived from the pre-steady state transients are plotted versus L-Serine concentration. k_obs 1(■), k_obs 2 (●). The protein concentration was 0.5 μM subunit. The lines are fits of the data to equation 5.

A total of 1,000 points were collected for each trace and at least 10 individual traces were averaged at each set of conditions. The binding transients were analyzed either with the Pro-Data Viewer fitting software provided with the instrument or with the data fitting function of KinTek Global Kinetic Explorer (16, 17) and fit to a single or a sum of exponential functions defined as(Eq. 1)where Y is the fluorescence intensity at time t, k_obs,i is the observed rate of the ith process with an amplitude of Ai and C is an offset value (18, 19). For NADH binding, linear plots of the observed rate (k_obs) versus ligand concentration were fit to,(Eq. 2)where k₁is the constant for the on rate and k₋₁ is the constant for the off rate for binding. For hyperbolic plots of the observed rates (k_obs) versus ligand concentration were fit to,(Eq. 3)where K_d is the dissociation constant for binding and k₂ and k₋₂ are the forward and reverse rate constants of a subsequent conformational change, respectively. Plots that display a decreasing k_obs with increasing ligand concentration indicated a rate limiting conformational change prior to binding (20, 21) and were fit to,(Eq. 4)where k₃ and k₋₃ are the forward and reverse rate constants of the conformational change and Kd* is the dissociation constant for ligand binding. Equations 3 and 4 can be combined into Scheme 2 (20) where ligand can bind either before or after the conformational change. The rate constants can be obtained by fitting to,(Eq. 5)where the rate and dissociation constants correspond to those in equations 3 and 4. With reference to Scheme 2, K_d = k₋₁/k₁ and K_d* = k₋₄/k₄.

The transients for NADH binding are shown for two time ranges, 0–1 s and 0–50 s (inset). The FRET signals are monitored at 420 nm after excitation at 295 nm. The enzyme concentration is 2.5 μM subunit. The transients are shown for 10, 15, 20, 35, and 45 μM NADH from lower to higher relative fluorescence. All data points are plotted for the 0–1 s range and every 50th data point are plotted for the 0–50 s range. The lines are fits of the data to equation 1 for 4 four exponentials.

The increase in signal at 340 nm is monitored with excitation at 295 nm in the presence of saturating levels of NADH (250 μM). Protein concentration is 2 μM subunit. The transients are shown for 10, 15, 40, 60, and 100 μM α-ketoglutarate from lower to higher relative fluorescence. The lines are fits of the data to equation 1 for a single exponential.

The amplitudes of the fits to equation 1 from the pre-steady-state transients that yielded the observed rates in Figure 3 are plotted versus α-ketoglutarate concentration. The amplitudes are determined in the presence of 0 (●), 2 (■), 3 (◆), and 4 μM (▲) L-serine. The lines are fits of the data to the equation for a rectangular hyperbola.

The decrease in signal at <340 nm is monitored with excitation at 295 nm in the presence of NADH (250 μM). Protein concentration is 0.5 μM subunit. The transients are shown for 3.5, 7.5, 10, 15, 20, 45, and 130 μM L̃serine from higher to lower relative fluorescence. The lines are fits of the data to equation 1 for 2 exponentials.

The amplitudes derived from fitting the pre-steady state transients of L-Serine binding to W139F-E360W PGDH are plotted; a_obs 1(■), a_obs 2 (●). The data were modeled to the theoretical distribution of bound species for a 4 Step sequential binding mechanism based on Adair constants of 15, 0.5, 3, and 120. The theoretical distribution of species shown represents E + EL₁ + EL3 (□) and EL₂ + EL₄ (○). In this case, L in the general equations refers to serine. The solid lines do not represent a fit to an equation, but simply connect the data points.

When α-ketoglutarate binding is measured in the absence of serine, a fluorescence signal cannot be detected unless NADH is already bound to the enzyme. In this case, a time dependent decrease in the 420 nm signal is observed as well as a complementary increase in fluorescence at 340 nm due to the enhancement of tryptophan fluorescence. When the signal is monitored as a function of α-ketoglutarate concentration, the transients fit best to 2 exponentials representing the 2 sets of sites consistent with that observed for NADH binding. The plots of k_obs versus α-ketoglutarate concentration in the absence of serine were hyperbolic and yielded a K_d for binding as well as forward and reverse rate constants for a subsequent rate limiting conformational change (13). When PGDH, combined with saturating concentrations of NADH and serine, was rapidly mixed with α-ketoglutarate, no signal was detected. Closer examination at serine concentrations in the range of the K_0.5 for serine inhibition (1–4 μM) produced a measurable signal (Figure 4) and revealed that the major effect of serine was to lower the amplitude of the fluorescence signal rather than substantially affect the observed rates. In fact, as the serine concentration is increased, data for one of the 2 sets of sites is lost since its amplitude was already at a low level. Figure 5 and 6 show the data for the major site and demonstrate that the observed rates as a function of α-ketoglutarate concentration are similar at all serine concentrations shown to that in the absence of serine (Figure 5). As the serine concentration is increased, the amplitude of the signal decreases (Figure 6), and at higher serine concentrations, the amplitude decreases to zero. This indicates that serine binding reduces the population of enzyme species capable of converting substrates to products. This results in the dead-end species composed of enzyme, serine, coenzyme, and substrate depicted in scheme 1 (ES·NADH·KG) and explains the basis for the V-type inhibition.