Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein

Abstract

The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ(1) (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s(-1) was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s(-1). The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483-491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ(1), a soluble analogue of ubiquinone, a rate constant of 5.4 s(-1) was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for k(cat) during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k(cat) and k(cat)/K(m) parameters.

Figure 1

Structure of the PRODH domain and FAD conformations of E. coli PutA in oxidized and reduced states. (A) The (βα)₈ barrel core structure of the PRODH domain is shown, highlighting the locations of the FAD cofactor (yellow) and THFA (green) bound at the si-face of FAD (PDB 1tiw) (B) Overlay of the conformational differences in the FAD cofactor between dithionite-reduced (PDB 2fzm) and THFA-bound (PDB 1tiw) PRODH domain structures. THFA-bound structure is colored yellow with THFA highlighted in green. Dithionite-reduced structure is colored teal and has hyposulfite bound in the proline binding site. Black dashed lines represent hydrogen bonds observed in both structures, red dashes are hydrogen bonds unique to the dithionite-reduced structure, and green dashes are hydrogen bonds only observed in the THFA-bound structure. Figures were generated with PyMOL.

Figure 2

Proline reductive half-reaction. (A) Oxidized PutA (27 μM after mixing) was mixed with 100 mM proline (after mixing) and spectral changes were monitored by stopped-flow multi-wavelength absorption using a photodiode array detector. The spectra shown were recorded at 0.025–10 sec after mixing. (Inset) Single wavelength trace at 451 nm fit to eq 1. (B) Observed rates constants k_obs1 and k_obs2 from the fit to eq 1 were plotted versus proline concentration and fit to eq 2 to yield k_max estimates of 38 s⁻¹ (k_obs1) and 11 s⁻¹ (k_obs2) and an apparent K_d value of 550 mM. (C) Global fit of the single wavelength traces at 451 nm for the reaction of PutA (10 μM after mixing) with 100 (green), 400 (yellow), 800 (blue), 1200 (black), and 1500 mM (red) proline (concentration after mixing) to the mechanism shown for the reductive half-reaction in Scheme 2. (D) FitSpace contour plots for the global fit of the data. The ratio of k₁ and k₋₁ was fixed by a K_d of 550 mM for proline. Rate constants and best-fit parameters are summarized in Table 1.

Figure 3

Stopped-flow mixing of THFA with oxidized PutA. (A) Oxidized PutA (31. 5 μM after mixing) was mixed with 200 mM THFA (after mixing) and followed by stopped-flow multi-wavelength absorption. (Inset) Single wavelength trace at 476 nm fit to a single exponential (eq 1). (B) k_obs plotted versus THFA concentration and fit to eq 3 to yield k_on= 368 M⁻¹s⁻¹ and k_off = 5 s⁻¹ (K_d=13.5 mM). (Inset) Amplitude from the fit to eq 3 plotted against the THFA concentration and fit to eq 4 to yield K_d= 6 mM. (C) Global fitting of the single wavelength traces at 476 nm from the rapid mixing of PutA (31. 5 μM after mixing) with 2 (red), 4 (green), 8 (yellow), 16 (blue), 32 (pink), 100 (cyan), and 200 mM (black) THFA (after mixing) to a one-step binding mechanism (E + THFA = E−THFA) where k_on and k_off are the association and dissociation rate constants for THFA binding, respectively. Fitting yielded k_on= 417 M⁻¹s⁻¹ and k_off = 4.2 s⁻¹ (K_d=10.2 mM). (D) FitSpace contour plot of the global fit of the data. Calculated lower and upper bounds are 375 – 469 M⁻¹s⁻¹ and 3.4 – 5.1 s⁻¹ for k_on and k_off, respectively.

Figure 4

Rapid reaction kinetics of the reverse reaction with P5C. (A) Reduced PutA (11.8 μM after mixing) was mixed with 3.33 mM P5C (after mixing) and followed by stopped-flow multi-wavelength absorption. (Inset) Reduced PutA (11.78 μM) was rapidly mixed with 0.5 (black), 1.5 (red), 2.5 (yellow), and 3.3 (green) mM P5C. The observed rate constants from single exponential fits of the data were plotted against P5C concentration and fit to a line (slope = 2.6 × 10⁻³ and intercept = 1.9 × 10⁻³). (B and C) Global fitting of the combined data from single wavelength traces at 451 nm for the reverse (B) and forward (C) reactions to the reductive half-reaction shown in Scheme 2. The progress curves in panel C are a replot of the data from Figure 2C. The global fitting in panels B and C also includes amplitudes from multiwavelength data for the proline reductive half-reaction with 100 and 400 mM proline as shown in the Supporting Information (Figure S4). (D) FitSpace contour plots for the global fitting of the combined data from the reverse and forward reactions. Rate constants and best-fit parameters are summarized in Table 1.

Figure 5

Stopped-flow kinetics of the oxidative half-reaction with CoQ₁. (A) Reduced PutA (15 μM after mixing) was mixed with 60 μM CoQ₁ (after mixing) and monitored by stopped-flow multi-wavelength absorption. (Inset) Single wavelength trace at 451 nm fit to eq 1. (B) k_obs1 and k_obs2 from fitting to eq 1 were plotted against CoQ₁ concentration and fit to eq 2 yielding a k_max of 7.5 s⁻¹ and K_d of 124 μM CoQ₁ for k_obs1, and a k_max of 4 s⁻¹ for k_obs2. (C) Global fit of the single wavelength traces at 451 nm for the reaction of reduced PutA with 100 (black), 150 (yellow), 200 (blue), and 250 μM (red) CoQ₁ (after mixing) fit to the mechanism shown for the oxidative half-reaction in Scheme 2. (D) FitSpace contour plot for the global fitting of the data to the mechanism. Rate constants and best-fit parameters are summarized in Table 1.

Figure 6

Double mixing experiment for the proline reductive half-reaction. (A) Reduced PutA (22.5 μM after first mixing) was mixed with 95 μM CoQ₁ (after first mixing) and allowed to age for 20 sec. After aging, PutA (11.25 μM after second mixing) was rapidly mixed with 100 mM proline (after mixing) in the second mixing chamber and monitored by stopped-flow multi-wavelength absorption. (B) Estimates of k_obs1 and k_obs2 from fitting to eq 1 were plotted against different proline concentrations and fit to eq 2 yielding a k_max of 59 s⁻¹ and K_d of 316 mM proline for k_obs1, and a k_max of 0.44 s⁻¹for k_obs2. (C) Single wavelength traces at 451 nm of the data at 25 (red), 100 (green), 400 (yellow), and 800 mM (black) proline (after mixing) were globally fit to the simulated mechanism for the reductive half-reaction in Scheme 2. (D) FitSpace contour plots for the fitted data to the mechanism. Rate constants and best-fit parameters are summarized in Table 1.

Scheme 1

Oxidation of proline to glutamate catalyzed by the PRODH and P5CDH domains of PutA.

Scheme 2

Mechanisms used to simulate the stopped-flow data for the reductive and oxidative half-reactions by Global Kinetic Explorer. E, oxidized PutA conformer 1; F, reduced PutA conformer 1; f, reduced PutA conformer 2; e, oxidized PutA conformer 2.