Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase

Abstract

5-Aminolevulinate synthase (ALAS), a pyridoxal-5'phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0-3.0 and 7.5-10.5) and temperature (20 and 37°C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH10.5 and pH9.5/37°C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420nm to 330nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH9.5/37°C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.

Keywords: Aminolevulinate synthase; Heme; Intrinsically disordered proteins; Molten globule; Protein folding; Pyridoxal-5′phosphate.

Figure 1

Crystal structure of R. capsulatus ALAS. The distribution of color indicates: in pink, amino acids with ionizable side groups whose theoretical pK_a is lower than 5 (i.e., Asp and Glu); in cyan, amino acids with ionizable side groups whose theoretical pK_a is greater than 5 (i.e., His, Cys, Tyr, Lys, and Arg); in yellow, amino acids with hydrophobic side chains (i.e., Ala, Val, Leu, Ile, Trp, Phe, and Met) and in white, all other amino acids (i.e., Ser, Thr, Asn, Gln, Pro, and Gly) [PDB 2BWO].

Figure 2

Effects of pH and temperature on the secondary structure of mALAS2. Far-UV CD spectra collected at (A) acidic pH and 20 °C, (B) acidic pH and 37 °C, (C) alkaline pH and 20 °C, (D) alkaline pH and 37 °C. The colors of the spectra are: pH 1 (pink), pH 2 (purple), pH 3 (gray), pH 7.5 (black), pH 8.5 (red), pH 9.5 (blue), and pH 10.5 (cyan).

Figure 3

Effects of pH and temperature on the tertiary structure of mALAS2. Near-UV CD spectra collected at different acidic and alkaline pH values at (A) 20 °C, and (B) 37 °C. (C) Spectra at 20 °C (solid) and 37 °C (dashed) at pH 7.5. (D) Spectra at 20 °C (solid) and 37 °C (dashed) at pH 9.5. The colors of the spectra are: pH 1 (pink), pH 2 (purple), pH 3 (gray), pH 7.5 (black), pH 8.5 (red), pH 9.5 (blue), and pH 10.5 (cyan).

Figure 4

Dependence of CD ellipticity difference (Δθ_280-305) with pH. The CD ellipticity difference (Δθ_280-305) was calculated by subtracting the degree of ellipticity at 280 nm from that at 305 nm. The symbols correspond to the spectra collected at different temperatures: 20 °C (squares) and 37 °C (triangles).

Figure 5

Effects of pH and temperature on the chiral active site environment of the mALAS2 internal aldimine. CD spectra in the 500-310 nm region collected at (A) alkaline pH and 20 °C, (B) alkaline pH and 37 °C, (C) acidic pH and 20 °C, (D) acidic pH and 37 °C. The colors of the spectra correspond to: pH 1 (pink), pH 2 (purple), pH 3 (gray), pH 7.5 (black), pH 8.5 (red), pH 9.5 (blue), and pH 10.5 (cyan).

Figure 6

Reaction between ANS and mALAS2 at different pH values at 20 or 37 °C. (A) Wavelength of emission maxima. (B) Fluorescence emission intensity. The colors correspond to: free ANS at 20 °C (black), free ANS at 37 °C (gray), ANS in the presence of mALAS2 at 20 °C (blue), and ANS in the presence of mALAS2 at 37 °C (pink).

Figure 7

Acrylamide quenching of intrinsic fluorescence of mALAS2. (A) pH 1.0 and 20 °C, (B) pH 7.5 and 20 °C, (C) pH 9.5 and 20 °C, (D) pH 1 and 37 °C, (E) pH 7.5 and 37 °C, (F) pH 9.5 and 37 °C.

Figure 8

Size distribution of mALAS2 at pH values of (A) pH 9.5, (B) pH 7.5, (C) pH 3.0, (D) pH 2.0, and (E) pH 1.0. All experiments were performed using 1 mg/ml protein and at 25 °C.

Figure 9

Binding of ALA to the alkaline molten globule state of ALAS restricts fluctuations in the tertiary structure. Near-UV CD spectra were collected at 37 °C with the reactants in 50 mM AMPSO, pH 9.5. Dashed and solid lines correspond to mALAS2 in the absence and presence of ALA, respectively.