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.

Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I

“…The K DðPLPÞ values for G41R-Ma, G41V-Ma, and G41R-Mi were found to be 1.5 AE 0.4, 0.55 AE 0.1, and 6.0 AE 0.5 μM, respectively, that are ∼6-, 2-, and 23-fold higher than that of AGT-Ma or AGT-Mi (2,8). Both AGT-Ma and AGT-Mi bind pyridoxamine 5 0 -phosphate (PMP) with a K DðPMPÞ value ≪0.1 μM, whereas even a prolonged time of incubation of the apo forms of G41 variants with PMP (up to 5 mM) does not result in a dichroic signal at 340 nm, typical of the AGT-PMP complex (2). Thus, G41 mutations exert a decrease in the PLP binding affinity and a dramatic reduction in the PMP binding affinity.…”

“…AGT-Ma and AGT-Mi were prepared as reported (2,8). Site-directed mutagenesis, expression, and purification of G41 variants in the C-terminal His-tagged form were performed using standard procedures as described in SI Text.…”

“…Each subunit includes a N-terminal extension (residues 1-21), a large N-terminal domain (residues 22-282) containing the PLP-binding lysine (K209), and a smaller C-terminal domain (residues 283-392) (1). Steady-state and pre-steady-state kinetic studies featuring the AGT transamination revealed high specificity for glyoxylate to glycine processing, consistent with a key role of AGT in glyoxylate detoxification (2). The human liver-specific AGT is localized in the peroxisomal matrix (3).…”

“…contributed equally to this work. 2 To whom correspondence should be addressed. E-mail: carla.borrivoltattorni@univr.it.…”

“…Currently, the way of treatment of this progressive and potentially fatal disease is poor, as the molecular bases of the effects of the various disease-associated point mutations are unknown. In the last years, a biochemical characterization of the pathogenic variants G82E-Ma and F152I-Mi allowed us to correlate the clinical and enzymatic phenotypes with the structural and functional properties of the corresponding variants (2,8).…”