The amino acid sequence of human hepatic peroxisomal L-alanine: glyoxylate aminotransferase 1 (AGTI) deduced from cDNA shows 78% sequence identity with that of rat mitochondrial AGTI, but lacks the N-terminal 22 amino acids (the putative mitochondrial targeting signal). In humans this signal appears to have been deleted during evolution by a point mutation of the initiation codon ATG to ATA. These data suggest that the targeting defect in primary hyperoxaluria type 1, in which AGT1 is diverted from the peroxisomes to the mitochondria, could be due to a point mutation that reintroduces all or part of the mitochondrial signal sequence.
In addition to the main transaminase reaction, the pyridoxal 5'-phosphate-dependent enzyme human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is able to catalyze the alpha,beta-elimination of beta-chloro-L-alanine with a catalytic efficiency similar to that of the physiological transaminase reaction with L-alanine. On the other hand, during the reaction of AGT with L-cysteine, changes in the coenzyme forms and analysis of the products reveal the occurrence of both beta-elimination and half-transamination of L-cysteine together with the pyruvate transamination. A mechanism in which a ketimine species is the common intermediate of half-transamination and beta-elimination of L-cysteine is proposed. L-cysteine partitions between these two reactions with a ratio of approximately 2.5. Rapid scanning stopped-flow and quench flow experiments permit the identification of reaction intermediates and the measurements of the kinetic parameters of L-cysteine half-transamination. The k(cat) of this reaction is 200- or 60-fold lower than that of L-alanine and L-serine, respectively. Conversely, L-cysteine binds to AGT with a binding affinity 30- and 200-fold higher than that of L-alanine and L-serine, respectively. This appears to be consistent with the calculated interaction energies of the L-cysteine, L-alanine and L-serine docked at the active site of AGT.
Alanine:glyoxylate aminotransferase (AGT) is a pyridoxal-phosphate (PLP)-dependent enzyme. Its deficiency causes the hereditary kidney stone disease primary hyperoxaluria type 1. AGT is a highly stable compact dimer and the first 21 residues of each subunit form an extension which wraps over the surface of the neighboring subunit. Naturally occurring and artificial amino acid replacements in this extension create changes in the functional properties of AGT in mammalian cells, including relocation of the enzyme from peroxisomes to mitochondria. In order to elucidate the structural and functional role of this N-terminal extension, we have analyzed the consequences of its removal using a variety of biochemical and cell biological methods. When expressed in Escherichia coli, the N-terminal deleted form of AGT showed the presence of the protein but in an insoluble form resulting in only a 10% soluble yield as compared to the full-length version. The purified soluble fraction showed reduced affinity for PLP and greatly reduced catalytic activity. Although maintaining a dimer form, it was highly prone to self-aggregation. When expressed in a mammalian cell line, the truncated construct was normally targeted to peroxisomes, where it formed large stable but catalytically inactive aggregates. These results suggest that the N-terminal extension plays an essential role in allowing AGT to attain its correct conformation and functional activity. The precise mechanism of this effect is still under investigation.
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.
In addition to the main transaminase reaction, the pyridoxal 5'-phosphate-dependent enzyme human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is able to catalyze the alpha,beta-elimination of beta-chloro-L-alanine with a catalytic efficiency similar to that of the physiological transaminase reaction with L-alanine. On the other hand, during the reaction of AGT with L-cysteine, changes in the coenzyme forms and analysis of the products reveal the occurrence of both beta-elimination and half-transamination of L-cysteine together with the pyruvate transamination. A mechanism in which a ketimine species is the common intermediate of half-transamination and beta-elimination of L-cysteine is proposed. L-cysteine partitions between these two reactions with a ratio of approximately 2.5. Rapid scanning stopped-flow and quench flow experiments permit the identification of reaction intermediates and the measurements of the kinetic parameters of L-cysteine half-transamination. The k(cat) of this reaction is 200- or 60-fold lower than that of L-alanine and L-serine, respectively. Conversely, L-cysteine binds to AGT with a binding affinity 30- and 200-fold higher than that of L-alanine and L-serine, respectively. This appears to be consistent with the calculated interaction energies of the L-cysteine, L-alanine and L-serine docked at the active site of AGT.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionHGNC
Although human alanine:glyoxylate aminotransferase (AGT) is imported into peroxisomes by a Pex5p-dependent pathway, the properties of its C-terminal tripeptide (KKL) are unlike those of any other type 1 peroxisomal targeting sequence (PTS1). We have previously suggested that AGT might possess ancillary targeting information that enables its unusual PTS1 to work. In this study, we have attempted to locate this information and to determine whether or not it is a characteristic of all vertebrate AGTs. Using the two-hybrid system, we show that human AGT interacts with human Pex5p in mammalian cells, but not yeast cells. Using (immuno)fluorescence microscopic analysis of the distribution of various constructs expressed in COS cells, we show the following. 1) The putative ancillary peroxisomal targeting information (PTS1A) in human AGT is located entirely within the smaller C-terminal structural domain of 110 amino acids, with the sequence between Val-324 and Ile-345 being the most likely candidate region. 2) The PTS1A is present in all mammalian AGTs studied (human, rat, guinea pig, rabbit, and cat), but not amphibian AGT (Xenopus). 3) The PTS1A is necessary for peroxisomal import of human, rabbit, and cat AGTs, but not rat and guinea pig AGTs. We speculate that the internal PTS1A of human AGT works in concert with the C-terminal PTS1 by interacting with Pex5p indirectly with the aid of a yet-to-be-identified mammal-specific adaptor molecule. This interaction might reshape the tetratricopeptide repeat domain allosterically, enabling it to accept KKL as a functional PTS1.
G41 is an interfacial residue located within the alpha-helix 34-42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (approximately 5,000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 --> R mutation causes a partial unwinding of the 34-42 alpha-helix and a displacement of the first 44 N-terminal residues including the active site loop 24-32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations.
J. Mol. Biol. 331, 643-652 (2003)[PubMed:12899834]
A deficiency of the liver-specific enzyme alanine:glyoxylate aminotransferase (AGT) is responsible for the potentially lethal hereditary kidney stone disease primary hyperoxaluria type 1 (PH1). Many of the mutations in the gene encoding AGT are associated with specific enzymatic phenotypes such as accelerated proteolysis (Ser205Pro), intra-peroxisomal aggregation (Gly41Arg), inhibition of pyridoxal phosphate binding and loss of catalytic activity (Gly82Glu), and peroxisome-to-mitochondrion mistargeting (Gly170Arg). Several mutations, including that responsible for AGT mistargeting, co-segregate and interact synergistically with a Pro11Leu polymorphism found at high frequency in the normal population. In order to gain further insights into the mechanistic link between genotype and enzymatic phenotype in PH1, we have determined the crystal structure of normal human AGT complexed to the competitive inhibitor amino-oxyacetic acid to 2.5A. Analysis of this structure allows the effects of these mutations and polymorphism to be rationalised in terms of AGT tertiary and quaternary conformation, and in particular it provides a possible explanation for the Pro11Leu-Gly170Arg synergism that leads to AGT mistargeting.
Interacting selectively and non-covalently with pyridoxal 5' phosphate, 3-hydroxy-5-(hydroxymethyl)-2-methyl4-pyridine carboxaldehyde 5' phosphate, the biologically active form of vitamin B6.
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.
Alanine:glyoxylate aminotransferase-1 (AGT) is a human liver peroxisomal enzyme whose deficiency results in, primary hyperoxaluria type 1 (PH1), a fatal metabolic disease. AGT requires a pyridoxal phosphate (PLP) co-factor in its active site. The AGT gene usually exists in one of two polymorphic forms, the major and minor alleles. We describe here an overexpression system for normal and mutant variants of human AGT in Escherichia coli BL21 (DE3) pLysS. We have extracted functional AGT from inclusion bodies using guanidine-HCl. Denaturation and re-folding of the overexpressed AGT after guanidine-HCl treatment produces high yields of biologically active protein and provides a strategy for generating an apoenzyme to investigate PLP-binding. K(M)s for PLP were determined by reconstitution of the apoenzyme. Successful folding was independent of the presence of PLP. The K(M) for PLP for minor allele AGT was significantly higher than that for major allele AGT. This decreased affinity could be attributed to I340M, a polymorphism associated with the minor allele. G170R, located on the minor allele and the most common PH1 mutation, had no effect on the affinity for PLP. PH1 mutations, G41V and G41R, showed enhanced activity after re-folding. We suggest that the renaturation/re-folding and reconstitution strategies provide an approach for studying the maturation of AGT under optimal conditions and in isolation from cellular quality control and chaperoning processes. Furthermore, our data show that mutations with serious consequences in vivo may not be inherently catalytically inactive and may be rescuable.
G41 is an interfacial residue located within the alpha-helix 34-42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (approximately 5,000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 --> R mutation causes a partial unwinding of the 34-42 alpha-helix and a displacement of the first 44 N-terminal residues including the active site loop 24-32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations.
Interacting selectively and non-covalently with one or more specific sites on a receptor molecule, a macromolecule that undergoes combination with a hormone, neurotransmitter, drug or intracellular messenger to initiate a change in cell function.
Evidence
1:
Inferred from Physical InteractionUniProtKB
Most proteins are targeted to the peroxisomal matrix by virtue of a peroxisomal targeting signal-1 (PTS1), a short carboxy-terminal sequence specifically recognized by the PTS1 receptor Pex5p. We had previously developed a model that allowed the estimation of the affinities of many PTS1 sequences within the human proteome for Pex5p that revealed a wide range of predicted affinities. We have now experimentally determined the affinities of the PTS1-containing peptides from 42 proteins from the human proteome for Pex5p and show that these range over 4 orders of magnitude. These affinities correlate reasonably well with the predicted values and are substantially more precise. In an attempt to provide a possible explanation for the wide range of PTS1-Pex5p affinities, we compared these affinities with mRNA levels (as a proxy for rates of protein production) of the genes encoding these proteins in 79 human tissues and cell types. We note that high affinity PTS1-Pex5p interactions tend to correspond to proteins encoded by genes expressed at relatively low levels, whereas lower affinity PTS1-Pex5p interactions tend to correspond to proteins encoded by genes exhibiting higher levels and wider ranges of expression. Further analysis revealed that these relationships are consistent with the notion that a relatively uniform pool of protein-Pex5p complexes is maintained for appropriate peroxisome assembly.
G41 is an interfacial residue located within the alpha-helix 34-42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (approximately 5,000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 --> R mutation causes a partial unwinding of the 34-42 alpha-helix and a displacement of the first 44 N-terminal residues including the active site loop 24-32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations.
In addition to the main transaminase reaction, the pyridoxal 5'-phosphate-dependent enzyme human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is able to catalyze the alpha,beta-elimination of beta-chloro-L-alanine with a catalytic efficiency similar to that of the physiological transaminase reaction with L-alanine. On the other hand, during the reaction of AGT with L-cysteine, changes in the coenzyme forms and analysis of the products reveal the occurrence of both beta-elimination and half-transamination of L-cysteine together with the pyruvate transamination. A mechanism in which a ketimine species is the common intermediate of half-transamination and beta-elimination of L-cysteine is proposed. L-cysteine partitions between these two reactions with a ratio of approximately 2.5. Rapid scanning stopped-flow and quench flow experiments permit the identification of reaction intermediates and the measurements of the kinetic parameters of L-cysteine half-transamination. The k(cat) of this reaction is 200- or 60-fold lower than that of L-alanine and L-serine, respectively. Conversely, L-cysteine binds to AGT with a binding affinity 30- and 200-fold higher than that of L-alanine and L-serine, respectively. This appears to be consistent with the calculated interaction energies of the L-cysteine, L-alanine and L-serine docked at the active site of AGT.
Alanine:glyoxylate aminotransferase (AGT) is a pyridoxal-phosphate (PLP)-dependent enzyme. Its deficiency causes the hereditary kidney stone disease primary hyperoxaluria type 1. AGT is a highly stable compact dimer and the first 21 residues of each subunit form an extension which wraps over the surface of the neighboring subunit. Naturally occurring and artificial amino acid replacements in this extension create changes in the functional properties of AGT in mammalian cells, including relocation of the enzyme from peroxisomes to mitochondria. In order to elucidate the structural and functional role of this N-terminal extension, we have analyzed the consequences of its removal using a variety of biochemical and cell biological methods. When expressed in Escherichia coli, the N-terminal deleted form of AGT showed the presence of the protein but in an insoluble form resulting in only a 10% soluble yield as compared to the full-length version. The purified soluble fraction showed reduced affinity for PLP and greatly reduced catalytic activity. Although maintaining a dimer form, it was highly prone to self-aggregation. When expressed in a mammalian cell line, the truncated construct was normally targeted to peroxisomes, where it formed large stable but catalytically inactive aggregates. These results suggest that the N-terminal extension plays an essential role in allowing AGT to attain its correct conformation and functional activity. The precise mechanism of this effect is still under investigation.
Alanine:glyoxylate aminotransferase (AGT) is a pyridoxal-phosphate (PLP)-dependent enzyme. Its deficiency causes the hereditary kidney stone disease primary hyperoxaluria type 1. AGT is a highly stable compact dimer and the first 21 residues of each subunit form an extension which wraps over the surface of the neighboring subunit. Naturally occurring and artificial amino acid replacements in this extension create changes in the functional properties of AGT in mammalian cells, including relocation of the enzyme from peroxisomes to mitochondria. In order to elucidate the structural and functional role of this N-terminal extension, we have analyzed the consequences of its removal using a variety of biochemical and cell biological methods. When expressed in Escherichia coli, the N-terminal deleted form of AGT showed the presence of the protein but in an insoluble form resulting in only a 10% soluble yield as compared to the full-length version. The purified soluble fraction showed reduced affinity for PLP and greatly reduced catalytic activity. Although maintaining a dimer form, it was highly prone to self-aggregation. When expressed in a mammalian cell line, the truncated construct was normally targeted to peroxisomes, where it formed large stable but catalytically inactive aggregates. These results suggest that the N-terminal extension plays an essential role in allowing AGT to attain its correct conformation and functional activity. The precise mechanism of this effect is still under investigation.
Activities of alanine:glyoxylate aminotransferase in the livers of two patients with primary hyperoxaluria type I were substantially lower than those found in five control human livers. Detailed subcellular fractionation of one of the hyperoxaluric livers, compared with a control liver, showed that there was a complete absence of peroxisomal alanine:glyoxylate aminotransferase. This enzyme deficiency explains most of the biochemical characteristics of the disease and means that primary hyperoxaluria type I should be added to the rather select list of peroxisomal disorders.
The chemical reactions and pathways resulting in the breakdown of L-alanine, the L-enantiomer of 2-aminopropanoic acid, i.e. (2S)-2-aminopropanoic acid.
Alanine:glyoxylate aminotransferase (AGT) is a pyridoxal-phosphate (PLP)-dependent enzyme. Its deficiency causes the hereditary kidney stone disease primary hyperoxaluria type 1. AGT is a highly stable compact dimer and the first 21 residues of each subunit form an extension which wraps over the surface of the neighboring subunit. Naturally occurring and artificial amino acid replacements in this extension create changes in the functional properties of AGT in mammalian cells, including relocation of the enzyme from peroxisomes to mitochondria. In order to elucidate the structural and functional role of this N-terminal extension, we have analyzed the consequences of its removal using a variety of biochemical and cell biological methods. When expressed in Escherichia coli, the N-terminal deleted form of AGT showed the presence of the protein but in an insoluble form resulting in only a 10% soluble yield as compared to the full-length version. The purified soluble fraction showed reduced affinity for PLP and greatly reduced catalytic activity. Although maintaining a dimer form, it was highly prone to self-aggregation. When expressed in a mammalian cell line, the truncated construct was normally targeted to peroxisomes, where it formed large stable but catalytically inactive aggregates. These results suggest that the N-terminal extension plays an essential role in allowing AGT to attain its correct conformation and functional activity. The precise mechanism of this effect is still under investigation.
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.
In addition to the main transaminase reaction, the pyridoxal 5'-phosphate-dependent enzyme human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is able to catalyze the alpha,beta-elimination of beta-chloro-L-alanine with a catalytic efficiency similar to that of the physiological transaminase reaction with L-alanine. On the other hand, during the reaction of AGT with L-cysteine, changes in the coenzyme forms and analysis of the products reveal the occurrence of both beta-elimination and half-transamination of L-cysteine together with the pyruvate transamination. A mechanism in which a ketimine species is the common intermediate of half-transamination and beta-elimination of L-cysteine is proposed. L-cysteine partitions between these two reactions with a ratio of approximately 2.5. Rapid scanning stopped-flow and quench flow experiments permit the identification of reaction intermediates and the measurements of the kinetic parameters of L-cysteine half-transamination. The k(cat) of this reaction is 200- or 60-fold lower than that of L-alanine and L-serine, respectively. Conversely, L-cysteine binds to AGT with a binding affinity 30- and 200-fold higher than that of L-alanine and L-serine, respectively. This appears to be consistent with the calculated interaction energies of the L-cysteine, L-alanine and L-serine docked at the active site of AGT.
The chemical reactions and pathways resulting in the breakdown of L-cysteine, the L-enantiomer of 2-amino-3-mercaptopropanoic acid, i.e. (2R)-2-amino-3-mercaptopropanoic acid.
In addition to the main transaminase reaction, the pyridoxal 5'-phosphate-dependent enzyme human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is able to catalyze the alpha,beta-elimination of beta-chloro-L-alanine with a catalytic efficiency similar to that of the physiological transaminase reaction with L-alanine. On the other hand, during the reaction of AGT with L-cysteine, changes in the coenzyme forms and analysis of the products reveal the occurrence of both beta-elimination and half-transamination of L-cysteine together with the pyruvate transamination. A mechanism in which a ketimine species is the common intermediate of half-transamination and beta-elimination of L-cysteine is proposed. L-cysteine partitions between these two reactions with a ratio of approximately 2.5. Rapid scanning stopped-flow and quench flow experiments permit the identification of reaction intermediates and the measurements of the kinetic parameters of L-cysteine half-transamination. The k(cat) of this reaction is 200- or 60-fold lower than that of L-alanine and L-serine, respectively. Conversely, L-cysteine binds to AGT with a binding affinity 30- and 200-fold higher than that of L-alanine and L-serine, respectively. This appears to be consistent with the calculated interaction energies of the L-cysteine, L-alanine and L-serine docked at the active site of AGT.
J. Cell Biol. 111, 2341-2351 (1990)[PubMed:1703535]
We have previously shown that in some patients with primary hyperoxaluria type 1 (PH1), disease is associated with mistargeting of the normally peroxisomal enzyme alanine/glyoxylate aminotransferase (AGT) to mitochondria (Danpure, C.J., P.J. Cooper, P.J. Wise, and P.R. Jennings. J. Cell Biol. 108:1345-1352). We have synthesized, amplified, cloned, and sequenced AGT cDNA from a PH1 patient with mitochondrial AGT (mAGT). This identified three point mutations that cause amino acid substitutions in the predicted AGT protein sequence. Using PCR and allele-specific oligonucleotide hybridization, a range of PH1 patients and controls were screened for these mutations. This revealed that all eight PH1 patients with mAGT carried at least one allele with the same three mutations. Two were homozygous for this allele and six were heterozygous. In at least three of the heterozygotes, it appeared that only the mutant allele was expressed. All three mutations were absent from PH1 patients lacking mAGT. One mutation encoding a Gly----Arg substitution at residue 170 was not found in any of the control individuals. However, the other two mutations, encoding Pro----Leu and Ile----Met substitutions at residues 11 and 340, respectively, cosegregated in the normal population at an allelic frequency of 5-10%. In an individual homozygous for this allele (substitutions at residues 11 and 340) only a small proportion of AGT appeared to be rerouted to mitochondria. It is suggested that the substitution at residue 11 generates an amphiphilic alpha-helix with characteristics similar to recognized mitochondrial targeting sequences, the full functional expression of which is dependent upon coexpression of the substitution at residue 170, which may induce defective peroxisomal import.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a cAMP (cyclic AMP, adenosine 3',5'-cyclophosphate) stimulus.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a glucocorticoid stimulus. Glucocorticoids are hormonal C21 corticosteroids synthesized from cholesterol with the ability to bind with the cortisol receptor and trigger similar effects. Glucocorticoids act primarily on carbohydrate and protein metabolism, and have anti-inflammatory effects.
IEAOrtholog Compara
Enzymatic activity
This protein acts as an enzyme. It is known to catalyze the following reactions
Enzyme that catalyzes the transfer of an alpha-amino group from an amino acid to an alpha-keto acid. The amino group is usually covalently bound by the prosthetic group pyridoxal phosphate.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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