Human wild-type alanine:glyoxylate aminotransferase and its naturally occurring G82E variant: functional properties and physiological implications

Abstract

Human hepatic peroxisomal AGT (alanine:glyoxylate aminotransferase) is a PLP (pyridoxal 5'-phosphate)-dependent enzyme whose deficiency causes primary hyperoxaluria Type I, a rare autosomal recessive disorder. To acquire experimental evidence for the physiological function of AGT, the K(eq),(overall) of the reaction, the steady-state kinetic parameters of the forward and reverse reactions, and the pre-steady-state kinetics of the half-reactions of the PLP form of AGT with L-alanine or glycine and the PMP (pyridoxamine 5'-phosphate) form with pyruvate or glyoxylate have been measured. The results indicate that the enzyme is highly specific for catalysing glyoxylate to glycine processing, thereby playing a key role in glyoxylate detoxification. Analysis of the reaction course also reveals that PMP remains bound to the enzyme during the catalytic cycle and that the AGT-PMP complex displays a reactivity towards oxo acids higher than that of apoAGT in the presence of PMP. These findings are tentatively related to possible subtle rearrangements at the active site also indicated by the putative binding mode of catalytic intermediates. Additionally, the catalytic and spectroscopic features of the naturally occurring G82E variant have been analysed. Although, like the wild-type, the G82E variant is able to bind 2 mol PLP/dimer, it exhibits a significant reduced affinity for PLP and even more for PMP compared with wild-type, and an altered conformational state of the bound PLP. The striking molecular defect of the mutant, consisting in the dramatic decrease of the overall catalytic activity (approximately 0.1% of that of normal AGT), appears to be related to the inability to undergo an efficient transaldimination of the PLP form of the enzyme with amino acids as well as an efficient conversion of AGT-PMP into AGT-PLP. Overall, careful biochemical analyses have allowed elucidation of the mechanism of action of AGT and the way in which the disease causing G82E mutation affects it.

Scheme 1. Reaction catalysed by AGT

Scheme 2. Mechanism of the reaction catalyzed by AGT

The double arrows represent transaldimination steps.

Figure 1. Absorption, CD and fluorescence spectral changes of AGT–PLP alone and in the presence of substrates or substrate analogue

Absorption (A) and CD (B) spectra of 7 μM AGT–PLP (–) and in the presence of 500 mM L-alanine (- - -), 500 mM glycine (········) or 500 mM D-alanine (-·-·-·). (C) Fluorescence emission spectra (excitation at 280 nm) of 1.7 μM AGT–PLP (–) and in the presence of 500 mM D-alanine (-·-·-·). In each case, the buffer was 100 mM potassium phosphate (pH 7.4).

Figure 2. Absorption, CD and fluorescence spectra of AGT–PMP alone and in the presence of glyoxylate

(A) Absorption spectra of 9 μM AGT–PMP (–) and in the presence of 5 mM glyoxylate (- - -). (B) CD spectra of AGT–PMP (–), AGT–PMP in the presence of 5 mM glyoxylate (- - -) and apoAGT after 15 h incubation with 100 μM PMP (········). (C) Fluorescence emission spectrum (excitation at 280 nm) of 1.6 μM apoAGT (········) and AGT–PMP (–). In each case, the buffer was 100 mM potassium phosphate pH 7.4

Figure 3. Time-dependent CD spectral changes occurring on incubation of apoAGT with PMP in the presence of glyoxylate

ApoAGT (········) (5 μM) was incubated with 100 μM PMP in the presence of 5 mM glyoxylate in 100 mM potassium phosphate buffer (pH 7.4) at 25 °C. CD spectra were recorded at 0.5, 7.5, 28, 51, 81, 120, 146, 171 and 184 s.

Figure 4. Modelling of the active site of the PMP- and L-alanine–PLP-bound forms of AGT

The AGT active site residues that are involved in cofactor binding are illustrated and labelled according to the sequence position of human AGT. The docked structures of AGT–PMP (magenta sticks) and AGT–PLP in complex with L-alanine (blue sticks) are shown. The structure of AGT–PLP complexed with aminooxyacetic acid (AOA) [5] is also shown for reference, as grey sticks. Oxygen atoms are coloured red, nitrogen atoms blue, and phosphorus orange. This figure was created using pyMOL [28].

Figure 5. Rapid scanning stopped-flow spectra obtained upon reaction of AGT–PLP with L-alanine at 25 °C

The first 20 spectra were collected at 0.001, 0.007, 0.013, 0.019, 0.025, 0.031, 0.037, 0.043, 0.049, 0.055, 0.061, 0.067, 0.073, 0.079, 0.085, 0.091, 0.097, 0.106, 0.118 and 0.13 s, the last 30 spectra were recorded from 0.142 to 0.85 s. AGT–PLP (7 μM) was mixed with L-alanine (100 mM) in 100 mM potassium phosphate buffer (pH 7.4) at 25 °C. The inset shows the dependence of the k_obs for the increase at 330 nm absorbance as a function of L-alanine concentration. The points are the experimental data, whereas the curve is from a fit to eqn (9).

Figure 6. Transient kinetic analysis of the reaction of AGT–PLP with glycine

Plot of the absorbance at 420 nm with time for the reaction of 7 μM AGT–PLP with 500 mM glycine. The dotted line represents a two-exponential fit. Inset: dependence of the rate constants (k_obs) for increase (■) or decrease (●) at 420 nm as a function of glycine concentration. The solid lines represent the data fitted to eqn (9).

Figure 7. Single-wavelength stopped-flow measurements of the reaction of AGT–PMP with glyoxylate at 25 °C

The reaction of AGT–PMP (7 μM) with various concentrations of glyoxylate in potassium phosphate buffer (pH 7.4) at 25 °C. Time courses at 420 nm are shown. The dotted lines are from a fit to eqn (8). The inset shows the dependence of the k_obs for the increase of the intensity at 420 nm as a function of glyoxylate concentration. The points shown are the experimental values, while the curve is from the data fitted to eqn (9).

Figure 8. Absorption and CD spectra of the G82E mutant

(A) Differential absorption spectra of 13 μM apomutant G82E in the presence of 500 μM PLP at the indicated times. (B) CD spectra of 10 μM apomutant G82E incubated for 90 min with 500 μM PLP before (–) and after (- - -) NaBH₄ reduction. In each case, the buffer was 100 mM potassium phosphate (pH 7.4).

Figure 9. PMP formation during the half-transamination reaction of L-alanine catalysed by the G82E mutant

L-Alanine (500 mM) was added to apoG82E mutant pre-incubated with 1 mM PLP (■); 1 mM PLP was added to a mixture containing apoG82E and 500 mM L-Alanine (○); 1 mM PLP and 500 mM L-alanine were incubated at 25 °C for 30 min, and then added to apomutant (▲). Aliquots were removed from the reaction mixtures at the indicated times and the PMP content was measured by HPLC as described in the Experimental section. In each case, the mutant concentration was 8.5 μM, and the buffer was 100 mM potassium phosphate (pH 7.4).