Molecular Insight into the Synergism between the Minor Allele of Human Liver Peroxisomal Alanine:Glyoxylate Aminotransferase and the F152I Mutation

Abstract

Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe(152) variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe(152) variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5'-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200-1400-fold lower. Thus, Phe(152) is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp(108), as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.

FIGURE 1.

Absorption and CD spectral changes upon addition of l-alanine to F152I-Mi. A, absorption spectra of 6 μm F152I-Mi (- - -) and of the enzyme plus 500 mm l-alanine (—) immediately (line 1) and after 0.6, 3, 8, 14, 26, 34, 60, 100, and 180 min (line 9). B, CD spectra of 5 μm F152I-Mi (- - -) and the enzyme plus 500 mm l-alanine (—) immediately (line 1) and after 8, 15, 23, 48, 68, 100, 125, and 206 min (line 9). In each case, the buffer used was 100 mm potassium phosphate, pH 7.4.

FIGURE 2.

Time course of the overall reaction of AGT-Ma, AGT-Mi, and Phe¹⁵² variants. AGT-Ma, AGT-Mi, or Phe¹⁵² variants at a concentration of 2.6 μm were incubated at 37 °C in 100 mm potassium phosphate buffer, pH 7.4. At the indicated times, aliquots were withdrawn and denatured. After removal of the precipitated protein by centrifugation, the supernatants were subjected to HPLC analysis as described under “Experimental Procedures.” A: •, PLP; ▪, PMP; □, glyoxylate; ○, pyruvate, for AGT-Ma; ▾, PLP; ▴, PMP; +, glyoxylate; ×, pyruvate, for AGT-Mi. B: □, PLP; ▪, PMP for F152I-Mi; ○, PLP; •, PMP for F152I-Ma; ⋄, PLP; ♦, PMP for F152A-Ma. The percentage of PLP or PMP is referred to the original PLP content of the enzymes. The curves represent the least-squares fit of a second-order rate equation to the data. The data shown are the means of three independent experiments. In each case, the standard error of the mean was less than 5%.

FIGURE 3.

Modeling of the active site of AGT-Ma and F152I variant in the PMP form. The putative location of Trp¹⁰⁸ at the active site of AGT-Ma and F152I variant in the PMP form is illustrated. Trp¹⁰⁸, Phe¹⁵², and PMP are represented as blue sticks in AGT-Ma, whereas Trp¹⁰⁸, Ile¹⁵², and PMP are represented as cyano sticks in F152I variant. Oxygen atoms are colored red, nitrogen atoms blue, and phosphorus orange. This figure was rendered using PyMOL (17).

FIGURE 4.

Single wavelength stopped-flow measurements of the reaction of F152A-Ma with l-alanine. The reaction of F152A-Ma (6 μm) with various concentrations of l-alanine in 100 mm potassium phosphate buffer, pH 7.4, at 25 °C. Time courses at 420 nm are shown. The dotted lines are from a fit to Equation 2. The inset shows the dependence of the k_obs for the decrease of the intensity at 420 nm as a function of l-alanine concentration. The points shown are the experimental values, whereas the curve is from the data fitted to Equation 3.

FIGURE 5.

Time-dependent loss of activity and integrated peak area of apoF152i-Mi upon incubation at 37 °C. ApoF152I-Mi was incubated at 1 (circles), 0.5 (up triangles), 0.25 μm (squares) at 37 °C in 100 mm potassium phosphate buffer, pH 7.4. Aliquots were withdrawn at the indicated times and assayed either for transaminase activity or for the integrated peak areas measured upon gel filtration on a size exclusion chromatography. For comparison, the transaminase activity and the integrated peak area of 0.25 μm apoAGT-Mi upon incubation at 37 °C (down triangles) are shown. Closed and open symbols represent percentage of transaminase activity and integrated peak area, respectively. The lines are drawn to guide the eye.