Biochemical and biophysical analysis of five disease-associated human adenylosuccinate lyase mutants

Abstract

Adenylosuccinate lyase (ASL), a catalyst of key reactions in purine biosynthesis, is normally a homotetramer in which three subunits contribute to each of four active sites. Human ASL deficiency is an inherited metabolic disease associated with autism and mental retardation. We have characterized five disease-associated ASL mutants: R194C and K246E are located at subunit interfaces, L311V is in the central helical region away from the active site, and R396C and R396H are at the entrance to the active site. The V(max) (at 25 degrees C) for R194C is comparable to that of WT, while those of L311V, R396C, R396H, and K246E are considerably reduced and affinity for adenylosuccinate is retained. The mutant enzymes have decreased positive cooperativity as compared to WT. K246E exists mainly as dimer or monomer, accounting for its negligible activity, whereas the other mutant enzymes are similar to WT in the predominance of tetramer. At 37 degrees C, the specific activity of WT and these mutant enzymes slowly decreases 30-40% with time and reaches a limiting specific activity without changing significantly the amount of tetramer. Mutant R194C is unique in being rapidly inactivated at the harsher temperature of 60 degrees C, indicating that it is the least stable enzyme in vitro. Conformational changes in the mutant enzymes are evident from protein fluorescence intensity at 25 degrees C and after incubation at 37 degrees C, which correlates with the loss of enzymatic activity. Thus, these disease-associated single mutations can yield enzyme with reduced activity either by affecting the active site or by perturbing the enzyme's structure and/or native conformation which are required for catalytic function.

FIGURE 1

(I) The crystal structure of the human ASL which was crystallized with the products and the substrates (PDB # = 2vd6) (8). Each individual subunit is color coded and one active site of four is designated by a circle. The substrate, SAMP, is shown in pink and located in the two upper active sites and the products AMP (green) and fumarate (gray) are shown in the two lower active sites. The positions of the amino acids L311, R194, and R396, (in white) and K246 (in light brown) are shown in the A subunit. (II) The positions of Trp residues are shown in the B subunit: W39 (in white) W43 (in purple) W45 (in maroon) W175 and W326 (in white). The substrate and the products were removed from the active sites for clarity.

FIGURE 2

Electrostatic partners which are in close proximity to the subunit interface residues, R194C (I) and K246E (II). The color of each residue corresponds to the color scheme of the ASL tetramer shown in Figure 1.

FIGURE 3

Specific activity (ν) vs [SAMP] plots of WT and the mutant enzymes. (A) Kinetic plots of WT and R194C mutant enzymes. (B) Kinetic plot of K246E. (C) Kinetic plot of L311V mutant enzyme. (D) Kinetic plots of R396 mutant enzymes.

FIGURE 4

(A) Time dependent thermal inactivation plots (E_t/E₀ vs time) of WT and the mutant enzymes at 37 °C. (B) The plots of ln [(E_t — E_∞) /(E₀ — E_∞)] vs time at 37 °C. The slope of each plot yields the k_obs of each enzyme. The E₀ and E_t, are the enzymatic velocities at time 0 and time t, respectively. The E_∞ is the limiting activity of each enzyme and E_∞ = f E₀ where f is the fraction of the limiting activity to the original activity of the enzyme.

FIGURE 5

Time dependent thermal inactivation plots (E_t/E₀ vs time) of WT and the mutant enzymes at 60 °C. It is assumed that E_t/E₀ goes to zero in calculating the rate constants for inactivation.

FIGURE 6

Gel filtration elution profiles of WT and mutant enzymes at 25 °C. These elution profiles were obtained by applying 500 μL of each protein (∼3 mg) to a Sephacryl 200HR column (1 × 88 cm) equilibrated with Enzyme Storage Buffer, pH 7.0. The molecular weight standards used are V₀, blue dextran (30 mL); Ferritin-440 kDa; Catalase-232 kDa; Yeast alcohol dehydrogenase-141 kDa; Albumin- 67kDa; Ovalbumin-43 kDa. (I) The elution profiles of WT, R194C and L311V enzymes. (II) The elution profiles of WT, R396H and R396C enzymes.

FIGURE 7

CD spectra of WT and mutant enzymes. These spectra were determined with the protein (∼0.34 mg/mL) in Enzyme Storage Buffer, pH 7.0. (A) The CD spectra of WT enzyme and of R194C and K246E mutant enzymes at 25 °C. (B) The CD spectra of WT enzyme and of L311V, R396C, and R396H mutant enzymes at 25 °C. (C) The CD spectra of WT enzyme at 37 °C.

FIGURE 8

The fluorescence spectra of WT and mutant enzymes at 25 °C and 37 °C were determined with the protein (∼0.33 mg/mL) in fresh Enzyme Storage Buffer, pH 7.0. (A) The fluorescence spectra of WT enzyme at 25 °C and 37 °C in the absence and presence of 6 M Gdn.HCl and the fluorescence spectra of free Trp in the Enzyme Storage Buffer, pH 7.0. (B) Limiting fluorescence intensities of WT and mutant enzymes at 25 °C (measured at 335 nm) and at 37 °C (measured at 337 nm).

FIGURE 9

(A) Time dependent fluorescence change at 37 °C (F_t/F₀ vs time) measured at 337 nm of WT and mutant enzymes. (B) The plots of ln [(F_t — F_∞) /(F₀ — F_∞)] vs time and the slope of each plot yields the k^F_obs of each enzyme. The F₀ and F_t, are the fluorescence intensities at time 0 and time t, respectively. The F_∞ is the limiting fluorescence intensity of each enzyme.