Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P17174 (aspartate aminotransferase)
 14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Arginine-386, the active-site residue of Escherichia coli aspartate aminotransferase (EC 2.6.1.1) that binds the substrate alpha-carboxylate, was replaced with tyrosine and phenylalanine by site-directed mutagenesis. This experiment was undertaken to elucidate the roles of particular enzyme-substrate interactions in triggering the substrate-induced conformational change in the enzyme. The activity and crystal structure of the resulting mutants were examined. The apparent second-order rate constants of both of these mutants are reduced by more than 5 orders of magnitude as compared to that of wild-type enzyme, though R386Y is slightly more active than R386F. The 2.5-A resolution structure of R386F in its native state was determined by using difference Fourier methods. The overall structure is very similar to that of the wild-type enzyme in the open conformation. The position of the Phe-386 side chain, however, appears to shift with respect to that of Arg-386 in the wild-type enzyme and to form new contacts with neighboring residues.
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PMID:Activity and structure of the active-site mutants R386Y and R386F of Escherichia coli aspartate aminotransferase. 199 8

The structure of Escherichia coli aspartate aminotransferase complex with the inhibitor 2-methylaspartate, and that of the mutant enzyme in which an arginine was substituted for a lysine residue thereby forming a Schiff base with the coenzyme pyridoxal 5'-phosphate, were determined at 2.5 A resolution, by the molecular replacement method using the known structure of pig cytosolic aspartate aminotransferase. The enzyme catalyzes the reversible transamination between L-aspartate and alpha-ketoglutarate, and forms a dimeric structure of two identical subunits. Each subunit comprises two domains, a small and a large one. Although, in general, the overall and secondary structure of E. coli enzyme are similar to those of higher animals, some differences of enzymatic action between the enzyme from E. coli and those from higher animals could be explained on the basis of the X-ray structures and molecular mechanics calculation based on them.
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PMID:Three-dimensional structures of aspartate aminotransferase from Escherichia coli and its mutant enzyme at 2.5 A resolution. 212 25

Substitution of a lysyl residue for Arg-386 of Escherichia coli aspartate aminotransferase resulted in an extensive decrease in Vmax values (0.8% with the aspartate-2-oxoglutarate pair and 0.2% with the glutamate-oxalacetate pair, compared with the corresponding values for the wild-type enzyme). Kinetic analysis of the four sets of half-reactions, the pyridoxal form of the enzyme with aspartate or glutamate and the pyridoxamine form with 2-oxoglutarate or oxalacetate, allowed us to define the independent effect of the mutation on the reactivity of each substrate. Decrease in the first order rate constant (kmax) was more pronounced in the reactions with five-carbon substrates (glutamate and 2-oxoglutarate) than in those with four-carbon substrates (aspartate and oxalacetate), while the increase in the apparent dissociation constant (Kd) was greater for four-carbon substrates than for five-carbon substrates. The decrease of overall catalytic efficiency as judged by the values, kmax/Kd, was more pronounced in the reactions with five-carbon substrates than in those with four-carbon substrates. Affinities for substrate analogs such as succinate, glutarate, 2-methylaspartate, and erythro-3-hydroxyaspartate, were also considerably decreased by the mutation of the enzyme. These findings indicate that the side chain of the lysyl residue, although it bears a positive charge similar to that of the arginyl residue, is not structurally adequate for the productive binding of a substrate during catalysis.
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PMID:Substitution of a lysyl residue for arginine 386 of Escherichia coli aspartate aminotransferase. 249 35

Activated macrophages convert L-arginine to citrulline and unstable nitrogen oxides that have cytotoxic properties. We recently have shown that the inhibition of protein synthesis in Kupffer cell (KC):hepatocyte (HC) coculture, following exposure to gram-negative bacterial endotoxin (lipopolysaccharide), is due to the metabolism of L-arginine by this cytotoxic pathway. Although this finding supports a role for activated KCs and the L-arginine-dependent mechanism in the HC dysfunction seen in sepsis, it and previous studies have failed to demonstrate direct damage to HCs by adjacent KCs. The current study was undertaken to determine if KCs exposed to lipopolysaccharide could directly damage HCs and, if so, whether the damage was dependent on the metabolism of L-arginine. By using the release of aspartate aminotransferase as a marker of HC damage, it was found that a significant aspartate aminotransferase release by KC:HC cocultures in response to lipopolysaccharide occurred only if L-arginine was present. In addition, requirements for significant aspartate aminotransferase release included KC:HC ratios of 7.5:1 or greater and L-arginine concentrations of 1 mmol or more. Although the KC-induced damage was mild, these results show that in vitro HC damage in KC:HC coculture does require the metabolism of L-arginine and supports a hypothesis that toxic L-arginine metabolites may contribute to liver cell damage in patients with sepsis.
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PMID:Kupffer cell cytotoxicity to hepatocytes in coculture requires L-arginine. 258 66

The early stages of insulin-dependent diabetes mellitus are characterized by a selective inability to secrete insulin in response to glucose, coupled to a better response to nonnutrient secretagogues. The deficient glucose response may be a result of the autoimmune process directed toward the beta-cells. Interleukin-1 (IL-1) has been suggested to be one possible mediator of immunological damage of the beta-cells. In the present study we characterized the sensitivity of beta-cells to different secretagogues after human recombinant IL-1 beta (rIL-1 beta) exposure. Furthermore, experiments were performed to clarify the biochemical mechanisms behind the defective insulin response observed in these islets. Rat pancreatic islets were isolated and kept in tissue culture (medium RPMI-1640 plus 10% calf serum) for 5 days. The islets were subsequently exposed to 60 pM human recombinant IL-1 beta during 48 h in the same culture conditions as above and examined immediately after IL-1 exposure. The rIL-1 beta-treated islets showed a marked reduction of glucose-stimulated insulin release. Stimulation with arginine plus different glucose concentrations, and leucine plus glutamine partially counteracted the rIL-1 beta-induced reduction of insulin release. The activities of the glycolytic enzymes hexokinase, glucokinase, and glyceraldehyde 3-phosphate dehydrogenase, were similar in control and IL-1-exposed islets. Treatment with IL-1 also did not impair the activities of NADH+- and NADPH+-dependent glutamate dehydrogenase, glutamate-aspartate transaminase, glutamate-alanine transaminase, citrate synthase, and NAD+-linked isocitrate dehydrogenase. The oxidation of D-[6-14C]glucose and L-[U-14C]leucine were decreased by 50% in IL-1-treated islets. Furthermore, there was a significant decrease in the ratios of [2-14C]pyruvate oxidation/[1-14C]pyruvate decarboxylation and L-[U-14C]leucine oxidation/L-[1-14C]leucine decarboxylation, indicating that IL-1 decreases the proportion of generated acetyl-coenzyme-A residues undergoing oxidation. However, in the presence of IL-1 there was a significant increase in L-[U-14C]glutamate oxidation. These combined observations suggest that exposure to IL-1 induces a preferential decrease in glucose-mediated insulin release and mitochondrial glucose metabolism. This mitochondrial dysfunction seems to reflect an impairment in proximal steps of the Krebs cycle. It is conceivable that the IL-1-induced suppression and shift in islet metabolism can be an explanation for the beta-cell insensitivity to glucose observed in the early phases of human and experimental insulin-dependent diabetes mellitus.
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PMID:Differential sensitivity to beta-cell secretagogues in cultured rat pancreatic islets exposed to human interleukin-1 beta. 266 6

X-ray crystallographic data have implicated Arg-292 as the residue responsible for the preferred side-chain substrate specificity of aspartate aminotransferase. It forms a salt bridge with the beta or gamma carboxylate group of the substrate [Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., & Christen, P. (1984) J. Mol. Biol. 174, 497-525]. In order to test this proposal and, in addition, to attempt to reverse the substrate charge specificity of this enzyme, Arg-292 has been converted to Asp-292 by site-directed mutagenesis. The activity (kcat/KM) of the mutant enzyme, R292D, toward the natural anionic substrates L-aspartate, L-glutamate, and alpha-ketoglutarate is depressed by over 5 orders of magnitude, whereas the activity toward the keto acid pyruvate and a number of aromatic and other neutral amino acids is reduced by only 2-9 fold. These results confirm the proposal that Arg-292 is critical for the rapid turnover of substrates bearing anionic side chains and show further that, apart from the desired alteration, no major perturbations of the remainder of the molecule have been made. The activity of R292D toward the cationic amino acids L-arginine, L-lysine, and L-ornithine is increased by 9-16-fold over that of wild type and the ratio (kcat/KM)cationic/(kcat/KM)anionic is in the range 2-40-fold for R292D, whereas this ratio has a range of [(0.3-6) x 10(-6)]-fold for wild type. Thus, the mutation has produced an inversion of the substrate charge specificity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of arginine-292 in the substrate specificity of aspartate aminotransferase as examined by site-directed mutagenesis. 316

The cytoplasmic isozyme of aspartate transaminase is inactivated by trypsin due to loss of a 19-residue peptide from the NH2-terminal region. A second peptide bond at Arg-25 is then cleaved by trypsin leaving a residual core protein, transaminase 26-412. Inactivation by trypsin resembles that for the mitochondrial enzyme (Sandmeier, E., and Christen, P. (1980) J. Biol. Chem. 255, 10284-10289), yet occurs 10 times faster for the cytoplasmic isozyme. In the mitochondrial enzyme, trypsin cleavage produces equal concentrations of proteins missing the first 26 and 31 amino acids. Sequence variation in the NH2-terminal regions can explain such differences. Specifically, the mitochondrial NH2 terminus has no trypsin-susceptible residue at position 19 and is stabilized by an electrostatic interaction between Asp-15 and Arg-292, whereas position 15 is a valyl residue in the cytoplasmic enzyme. Calorimetric data reveal both a decreased transition temperature (Td) and enthalpy (delta Hd) of denaturation in transaminases 20-412 and 26-412. Interaction of substrates with the active site chromophore and differential scanning calorimetry (DSC) reveal that catalytically inactive transaminases 20-412 and 26-412 can bind amino acid substrates and produce spectroscopically detectable conversion of the pyridoxal to the pyridoxamine form of the protein. By contrast, substrate analogs only form enzymatic Michaelis-type complexes.
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PMID:Selective tryptic cleavage of native cytoplasmic aspartate transaminase holoenzyme. 636 6

Inactivation of the beta 2 subunit and of the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of one arginyl residue per beta monomer. The substrate L-serine protects the holo beta 2 subunit and the holo alpha 2 beta 2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by both the holo beta 2 subunit and the holo alpha 2 beta 2 complex indicate that L-serine and phenylglyoxal both bind to the same essential arginyl residue in the holo beta 2 subunit. The apo beta 2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by either pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo beta 2 subunit binds pyridoxal phosphate and the alpha subunit but cannot bind L-serine or L-tryptophan. We conclude that the alpha-carboxyl group of L-serine and not the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the beta 2 subunit. The specific arginyl residue in the beta 2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal has been identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains three other basic residues (lysine-137, arginine-141, and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148, and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium, and yeast supports a functional role for these three residues in anion binding. The location and role of the active-site arginyl residues in the beta 2 subunit and in two other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.
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PMID:L-serine binds to arginine-148 of the beta 2 subunit of Escherichia coli tryptophan synthase. 641 46

Reaction of the pyridoxal form of cytosolic aspartate aminotransferase from pig heart with 1,2-cyclohexanedione or other alpha-dicarbonyls led to a progressive decrease in the enzymic activity toward natural dicarboxylic substrates. The inactivation was prevented by the presence of dicarboxylic substrate analogs. The dependence of the inactivation rate on the cyclohexanedione concentration indicated that the modifying reagent forms a dissociable complex with the enzyme prior to the inactivation. These saturation kinetics were observed also with other alpha-dicarbonyls tested. The inactivation was fully accounted for by the modification of a single arginine residue per monomeric unit of the enzyme. Activities for alpha, beta-elimination reaction with 3-chloro-L-alanine and transamination with L-alanine did not decrease but appeared to increase considerably with the progress of the arginine modification. In these aberrant reactions, affinity for the monocarboxylic substrates was higher with the modified enzyme than with the native unmodified enzyme. Glutamate or aspartate was still capable of reacting with the pyridoxal form of the extensively modified enzyme to produce the pyridoxamine form at a rate comparable to that of the reaction with 3-chloro-L-alanine or L-alanine. Succinate, glutarate, maleate, 2-methylaspartate or erythro-3-hydroxy-aspartate which bind strongly to the native enzyme and thus acts as potent inhibitors in the reactions with monocarboxylic substrates did not exhibit any appreciable inhibitory effect on these reactions catalyzed by the arginine-modified enzyme. Proton NMR spectroscopy demonstrated that succinate strongly interacts with the native enzyme to generate substantial changes in the enzyme spectra whereas there was no such evidence for the specific interaction with this dicarboxylate with the arginine-modified enzyme.
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PMID:A critical arginine residue in cytosolic aspartate aminotransferase from pig heart. 707 57

Mitochondrial aspartate aminotransferase is inactivated by dicarbonyl reagents selectively modifying arginyl residues. Treatment with phenylglyoxal inactivates the enzyme with concomitant modification of 2.7 mol of arginyl residues/mol of subunit. If the reaction is performed in the presence of the transaminating substrate pair aspartate/oxalacetate, only 1.3 mol of arginyl residues/mol of subunit are labeled and the enzymic activity remains at 75% of the original value. One particular residue, identified by peptide analysis as Arg 292, is completely protected against modification in the presence of the substrate pair, indicating a role of its guanidinium group in substrate binding. On the basis of x-ray crystallographic studies of the complex of apoenzyme with N-(5'-phosphopyridoxyl)-aspartate (minus pyridoxal form of the enzyme), Arg 292 has been proposed as the binding site of the distal carboxylate group (Ford, G. C., Eichele, G., and Jansonius, J. N. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2559-2563). The enzyme with blocked Arg 292 is not completely inactive, and its molecular activity toward dicarboxylic substrates is of the same order of magnitude as that of the native enzyme toward alanine, which is 10(5) times lower than that toward dicarboxylic substrates. The activity toward alanine is unchanged but the rate-enhancing effect of formate on the transamination of alanine is impaired. Formate is assumed to occupy the binding site of the distal carboxylate group (Morino, Y., Osman, A. M., and Okamoto, M. (1974) J. Biol. Chem. 249, 6684-6692). Apparently, the interaction of the distal carboxylate group of the substrate with Arg 292 underlies not only the binding specificity but also the kinetic specificity of aspartate aminotransferase for dicarboxylic substrates.
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PMID:Chemical modification of a functional arginyl residue (Arg 292) of mitochondrial aspartate aminotransferase. Identification as the binding site for the distal carboxylate group of the substrate. 708


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